Taxon-specific zeta -crystallin in Japanese tree frog (Hyla japonica) lens

The present study demonstrated that the 38-kDa protein, instead of rho-crystallin (36 kDa), is expressed taxon specifically in the lens of Japanese tree frog (Hyla japonica). The 38-kDa protein was distinguished from rho-crystallin expressed in the lenses of bullfrog (Rana catesbeiana) and European common frog (Rana temporaria) immunochemically. Although the N terminus of the 38-kDa protein was blocked, the analyses of partial amino acid sequences showed that the protein was zeta-crystallin. Analysis of cDNA sequence encoding zeta-crystallin of the tree frog lens demonstrated that the deduced protein consisted of 329 amino acids including initial methionine and having 62.2 and 62.9% identity with zeta-crystallin of camel and guinea pig lenses, respectively. The molecular mass of the deduced structure was calculated to be 35,564 Da. zeta-Crystallin of the tree frog lens exhibited the intrinsic enzymatic activity of quinone reductase (EC, NADPH:quinone oxidoreductase). The crystallin specifically catalyzed the reduction of 9,10-phenanthrenequinone (Km, 42 microm) using NADPH (Km, 60 microm) as a cofactor. The enzymatic activity was inhibited by dicumarol, anti-coagulant drug, with IC50 of 4 microm. On gel filtration chromatography, the crystallin was recovered as 150-kDa molecular mass complex, indicating that the crystallin was homotetramer consisting of 38-kDa subunits. The crystallin gene was expressed specifically in the lens. These results show that taxon-specific crystallins such as zeta- and rho-crystallins may be available for the biochemical discrimination of Hyla- and Rana groups among frogs.     

Light- and electron-microscopic study of the endolymphatic sac of the tree frog,Hyla arborea japonica

Summary The endolymphatic sac of the tree frog and its crystals were observed by light- and electron microscopy. Scanning electron microscopy revealed that the crystals have a faceted body and two pointed ends. Light- and transmission electron microscopy revealed that the endolymphatic sac is composed of many small chambers. In their lumina, numerous ghosts of crystals that resulted from decalcification were observed. The ghosts were demarcated by a linear dense material or embedded in a flocculent substance. The epithelium of the endolymphatic sac is simple squamous or cuboidal and peculiar cytoplasmic granules are found in most cells. The granules are surrounded by a limiting membrane and have varying electron density. Some granules contain a core and/or tubular structures. Vacuoles containing large ghosts are also found in the epithelial cells. These ghosts were quite similar to those in the lumen and sometimes coexist with cell debris. The fine structure of the endolymphatic sac and its crystals is discussed.     

Effects of calcium preloading on the growth of calcium carbonate crystals in the endolymphatic sac of the tree frog,Hyla arbores japonica

Summary Tree frogs, either with or without calcium chloride preloading, were maintained in a 0.8% strontium chloride solution for 1 week, then studied by X-ray microanalysis and scanning electron microscopy to determine the distribution of incorporated strontium in the endolymphatic crystals. In the absence of calcium preloading, strontium was detected on every surface of all the crystals, but after calcium preloading for 5 or 7 weeks, strontium incorporation was partially or completely inhibited, suggesting that an inhibition of the growth of the endolymphatic crystals had taken place in these preloaded specimens.     

Sexually dimorphic muscles in the forelimb of the Japanese toad,Bufo japonicus

During the breeding season, male anurans display clasping behavior by holding females with their forelimbs. This behavior is peculiar to males, and may require specializations in forelimb musculature. The present study revealed that five kinds of forelimb muscles were heavier in the male Japanese toad than in the female: the flexor carpi radialis (FCR), the flexor antibrachii medialis caput superius (FAMsup), the abductor indicis longus (AIL), the extensor carpi radialis caput superius (ECRsup), and the flexor antibrachii lateralis superficialis caput superius (FALSsup). In addition, one breast muscle, the coracoradialis (CR), was also heavier in males than in females. A quantitative analysis of muscle fibers processed for myosin ATPase activity showed that, in such “sexually dimorphic muscles” of the female, both fast (twitch) and slow (tonic) muscle fibers were of smaller diameter than in other forelimb muscles of both sexes (all male muscles plus “nondimorphic muscles” of the female). Moreover, both types of fibers were less numerous than in the corresponding muscles of the male. These results suggest that the “sexually dimorphic muscles” are used especially for clasping by the male and are degenerative or subnormal in the female. Slow muscle fibers were neither peculiar to, nor abundant in, these clasping muscles, although they may well be necessary for tonic and prolonged contractions of the forelimb muscles during clasping. The mechanism of sexual dimorphism may be a direct action of androgens on clasping muscles or an indirect action on clasping muscles via the innervating motoneurons.     

Neuroepithelial bodies in the lung of the tree frog,Hyla arborea L.

Summary The endocrine-like cells (ELC), which together with nerve endings form the neuroepithelial bodies, are located on primary and secondary septa in the non-ciliated epithelium of the lung of Hyla arborea. ELC protrude markedly toward the lumen of the lung and are surrounded by pneumocytes, which separate ELC from the lumen by thin cytoplasmic processes. ELC possess a light cytoplasm containing two types of granules: (i) numerous small granules, 50–110 nm in diameter, and (ii) single large granules, 290–860 nm in diameter. Numerous nerve fibers, often forming synaptic junctions, can be observed in contact with ELC.This study was supported by a grant No. 476/II from the Polish Academy of Sciences     

Hormone induced chromatophore changes in the European tree frog, Hyla arborea, in vitro

The colours of the European tree frog, Hvlu urhorea , depend on three types of chromatophores: in dermo-epidermal direction melanophores, iridophores, and xanthophores. The ability ofthis species to assume a wide range ofcolours implies that very extensive changes in the chromatophores take place, which in turn require control by several regulating factors. The responses of the different chromatophore types to hormones with known melanophore-affecting abilities (α-MSH, β-MSH, ACTH, melatonin) were tested in an in vitro system (freshly explanted skin) using reflectance microspectrophotometry, light microscopy and time-lapse cinemicrography.
α-MSH, β-MSH and ACTH all induce a rapid dispersion of melanosomes during the 10 min after addition. The degree of pigment dispersion induced by ACTH is slightly less than after stimulation with α-MSH or β-MSH.
The iridophores react to MSH or ACTH treatment with a contraction of the entire cell (causing a reduction in reflecting area), and a change in orientation of the platelets, causing a decrease in selective reflectance. The iridophores appear to be especially sensitive to ACTH. A very striking feature of the iridophores when studied with time-lapse cinematography is their strong pulsations (approx. once per minute).
The xanthophores react to MSH and ACTH with a contraction. These cells appear to be sensitive to β-MSH in particular.
Melatonin strongly counteracts the effects of α-MSH, β-MSH and ACTH on all chromatophores.
These studies confirm the dynamic nature not only of the melanophores, but also of the iridophores and xanthophores, as pointed out by Schmidt (1920) and Nielsen (1978a). Furthermore the differences in the time course of the stimulation of the different types of chromatophores by various hormones may provide an experimental basis for the explanation of colour changes in Hyfa arboreu.     

The digital pads of the tree frog, Hyla cinerea. I. The epidermis

As the digital pad cells grow from the germinal epithelium, the desmosomes on the distal surface oF tile cells become aligned and form pegs filled with tonofilaments on the surface of the outer row of cells. The outer pad cells are separated from each other distally, thus the cell and the pegs form two sizes of protrusions in series on the pad surface which can fit into substratum/irregularities. A dense material, apparently derived from transforming bodies in the cells, coats the plasma membrane of the surface cells, presumably to strengthen them.     

The digital pads of the tree frog, Hyla cinerea. II. The mucous glands

The mucous glands consist of a single row of cells surrounded by smooth muscle. The cells are attached at their apical and basal regions and only cytoplasmic projections loosely link the lateral aspects of adjacent cells. Material accumulates in the cisternae of the rough endoplasmic reticulum, appears to form into dense granules in the Golgi apparatus, and then before being secreted, undergoes chemical and morphological alterations. Since some glands secrete onto the dorsal epidermis of the digits, the mucous is believed to function as a wetting agent for the skin as well as an aid to climbing.     

Seasonal variation in freeze tolerance and ice content of the tree frog Hyla versicolor

Freeze tolerance and ice content of Hyla versicolor showed pronounced variation between summer (June) and winter (December). Summer frogs survived freezing at -3 degrees C for up to 9 hr and ice accumulation up to 50% of their total body water. A time course of ice formation indicated that an equilibrium level was reached in approximately 15 hr. Thus, the lethal ice content was less than the equilibrium ice content for these conditions (63.1%). A second group was induced to enter an overwintering condition by holding them through the summer and then subjecting them to a progressive reduction in temperature and photoperiod for 2 months. These frogs survived freezing for 48 hr at -3 degrees C. Their equilibrium ice content at this temperature was significantly lower (52.5%) than comparably treated summer animals. In the winter acclimatized group, frozen frogs had substantially higher blood glucose levels than unfrozen frogs (22.7 mumol/ml vs. 1.33 mumol/ml), but glycerol levels were not elevated after freezing. Freezing frogs conditioned for overwintering at -7 degrees C resulted in a higher equilibrium ice content (62.6%), but none survived. It is evident that in preparation for overwintering, frogs reduce the amount of ice formed at a given subzero temperature, but there is little indication of a substantial change in the total amount of ice tolerated.     

Isolation and characterization of simple sequence repeat loci in the gray tree frog, Hyla chrysoscelis.

A gray tree frog (Hyla chrysoscelis) genomic library was constructed and characterized with regard to the incidence and complexity of simple sequence repeat (SSR) loci. The partial genomic library, containing approximately 10,000 clones with an average-sized insert of 350 bp, was screened with six SSR repeat oligonucleotides (AC, AG, ACG, AGC, AAC, and AAG). Screening identified 31 unique positive clones containing 41 SSR loci. Sequences of tandemly arrayed dinucleotide repeats were more common (36 of 41) than trinucleotide repeats. Twenty-six loci were identified using the AC dinucleotide probe, while 7 loci were identified using the AG dinucleotide probe. An additional 3 AT dinucleotide loci were serendipitously identified. The AT repeats generally comprised the longest dinucleotide repeat loci. The SSR repeat loci reported here should provide potent markers for identity, parentage, and short-lineage determinations in large-scale experiments using gray tree frogs.     

Isolation and characterization of microsatellite loci for the European tree frog (Hyla arborea)

We developed 11 new microsatellite markers for the European tree frog (Hyla arborea), and tested patterns of polymorphism in 54 adults (27 males and 27 females) from two ponds close to Lausanne (Western Switzerland). One marker was sex linked and two pairs displayed linkage disequilibrium. Comparisons of allele numbers with heterozygosity values support a stepwise-mutation model at neutral equilibrium, with mutation rates spanning nearly two orders of magnitude. These markers will prove useful for population genetic studies and fine-scale investigations requiring genetic assignment techniques.     

Paracosmocercella rosettae n. gen. et n. sp. (Nematoda: Cosmocercoidea: Cosmocercidae) collected from the Japanese tree frog, Hyla japonica (Anura: Hylidae), in Japan

A new genus, Paracosmocercella (Nematoda: Cosmocercoidea: Cosmocercidae), is proposed with monotypic Paracosmocercella rosettae n. sp. from the Japanese tree frog, Hyla japonica Günther, 1859, in Oita, Kyushu Island, Japan. Paracosmocercella resembles Cosmocercella and Cosmocercoides by having 2 rows of rosette papillae without plectanes ventrally in preanal region of male, but it is readily distinguished from Cosmocercella by lacking clear vesicle supporting rosette papillae and from Cosmocercoides by lacking rosette papillae in perianal region and having large-sized eggs, which hatch in uterus.     

The effects of five organophosphorus insecticides on thermal stress in tadpoles of the Pacific tree frog, Hyla regilla

Three week old tadpoles of Hyla regilla were exposed to the insecticides temephos, fenthion, methyl parathion, chlorpyrifos, and malathion for 24 h at concentrations less than and at field applied rates as recommended for mosquito control in California. The thermal tolerance was significantly lowered by all chemicals and concentrations tested. Chlorpyrifos and methyl parathion were the most active compounds tested whereas malathion was the least toxic to H regilla tadpoles. Activity was depressed, in comparison to control tadpoles, by the chemicals methyl parathion (100 ppb) and malathion (500 ppb).     

Genetic diversity and fitness in peripheral and central populations of the European tree frog Hyla arborea

Genetic diversity is expected to decrease in small and isolated populations as a consequence of founder effects, bottlenecks, inbreeding and genetic drift. In this study we analyse temporal and spatial effects on genetic variation and progeny viability of the European tree frog (Hyla arborea) at two scales. First, the Swedish distribution has been isolated from the continental distribution for more than 8000 thousand years, and secondly, within Sweden, recent habitat alterations that have taken place during this century have increased isolation between local populations. Genetic variation and progeny survival in relation to isolation was studied within the entire Swedish distribution of the tree frog. Allozyme electrophoresis analysis of froglets, sampled across the Swedish distribution, revealed a low overall genetic variation (1.06 alleles/locus) at the protein level in comparison with continental populations (1.54-1.68 alleles/locus). However, egg hatchability (97%) and early larval survival (95%) were not lower than in other parts of the tree frog distribution or in other anuran species. Within the Swedish distribution, early larval survival was lower in isolated breeding ponds than in more central ones. However, no differences in genetic variation were found in relation to isolation. Polymorphism was detected only at a single locus, and was restricted geographically to the eastern part of the Swedish distribution. Bottlenecks due to climatic changes and fragmentation of suitable habitat (primarily natural pastures with ponds) are suggested as possible causes of the low genetic diversity of the Swedish tree frog population.     

Season and testosterone affect contractile properties of fast calling muscles in the gray tree frog Hyla chrysoscelis

In anurans, circulating levels of androgens influence certain secondary sexual characteristics that are expressed only during the breeding season. We studied the contractile properties of external oblique muscles (used to power sound production) in a species of North American gray tree frog, Hyla chrysoscelis, during the breeding season and also in testosterone-treated captive males and females after the breeding season. Compared with the muscles of breeding-season males, the trunk muscles of postbreeding-season males have 50% less mass, 60% longer twitches, and 40% slower shortening velocities. Testosterone levels similar to those found in breeding-season male hylid frogs restore the contractile speed and mass of male trunk muscles and also convert the small slow trunk muscles of females into larger fast-contracting muscles. We conclude that androgens likely play a key role in altering the contractile properties of these muscles in males during the annual cycle, allowing them to operate in the breeding season at the frequencies required to produce the characteristic rapidly pulsed calls of this species. Females as well as nonbreeding-season males do not produce advertising calls, and therefore the slower muscles found in these animals may allow more economic operation of these muscles. The effects of testosterone on female trunk muscles indicate the potential of this hormone in contributing to the sexual dimorphism in size and contractile properties of these muscles, but this dimorphism is likely due to the interaction of more than one hormone.     

Individual, nightly, and seasonal variation in calling behavior of the gray tree frog, Hyla versicolor: implications for energy expenditure

We investigated individual, nightly, and seasonal variationin calling behavior of a population of gray tree frogs (Hylaversicolor) from Connecticut, USA. Repeated recordings of individualmales on seven nights revealed significant differences amongmales in calling rate on all but one night and differences innumber of pulses per call and number of pulses produced perhour (pulse effort) on four nights. Most males reduced callingactivity late at night (after 2230 h), but some maintained arelatively steady rate of call production before dropping outof the chorus. Data collected for 26 individuals recorded onthree or more nights throughout the breeding season revealedsignificant differences among males in calling rate, numberof pulses per call, and pulse effort, but repeatabilities forall three variables were low (0.17, 0.35, and 0.12, respectively).The highest repeatability was for number of pulses per call,a variable strongly influenced by proximity to calling neighbors,probably because males often interacted with neighbors at similardistances on several successive nights. Males tended to reducethe number of pulses per call as the season progressed and thedistance between neighbors decreased, but they showed no clearseasonal change in calling rate or pulse effort. There was asubstantial seasonal decline in the number of hours of chorusactivity, resulting in a median decrease of 43% in nightly energyexpenditure by calling males.     

Tanycyte absorption affected by the hypothalamic deafferentation in Japanese quail,Coturnix coturnix japonica

In Japanese quail, Coturnix coturnix japonica the tanycytes of the median eminence absorbed peroxidase injected into the third ventricle. The number of tanycytes showing peroxidase reaction was greater in the posterior median eminence than in the anterior median eminence. Following hypothalamic deafferentation, the tanycyte absorption was augmented both in the posterior and anterior median eminence. These findings suggest that axons of some neurons, which have inhibitory action on the tanycyte absorption, were transected by deafferentation resulting in augmentation of tanycyte absorption. A considerable number of ependymal cells lining the upper portion of the third ventricle and those of the pars nervosa also absorbed peroxidase. In birds with a deafferented hypothalamus, photostimulated ovarian growth was completely inhibited.     

Fixed green and brown color morphs and a novel color-changing morph of the Pacific tree frog Hyla regilla

Pacific tree frogs Hyla regilla are typically either green or brown in dorsal coloration. The frequency of green and brown individuals is known to fluctuate seasonally. Previous investigators have generally assumed that the green and brown body colors represent a "fixed" polymorphism and that seasonal changes in the proportion of the two body colors are a consequence of differential survival of the two color morphs. Here we report that, in addition to the "fixed" (i.e., non-color-changing) green and brown morphs of H. regilla, there are some individuals that can change hue between green and brown. The distribution of color-change ability in our study population is bimodal, suggesting that "color changers" are a distinct morph rather than one extreme of a continuous distribution of color-change ability. Our findings suggest that background brightness, not hue, triggers color change in the newly discovered morph and that this change requires days to weeks to occur. Such slow color change is not well suited for making short-term changes in color as a frog moves between differently colored substrates. Rather, seasonal changes in habitat characteristics and/or microhabitat use are likely to maintain color-change ability. Color polymorphism and color-change ability appear to represent alternative responses to divergent selection for crypsis in a heterogeneous, seasonally variable environment.     

Development in Japanese macaques (Macaca fuscata): Sexually dimorphic behavior during the first year of life

We have documented several sexually dimorphic patterns of behavior that develop during the first year of life in infant Japanese macaques and their mothers. Mothers treated their infants differently by sex—mothers of males broke contact with them and retrieved them more frequently than did mothers of females. And mothers of male infants moved more frequently than did mothers of female infants. Male infants played more, played in larger groups, and mounted more frequently; female infants groomed and spent more time close to other monkeys in larger social groups than did males. Female infants were also punished by other group members more frequently than were male infants. We conclude that male and female Japanese macaque infants receive differential treatment early in life by both their own mothers and other animals, and males and females in turn treat their mothers and other animals differently. There appears to be a reciprocal relationship between the behavior of infants, mothers and other social partners that contributes to the development of sexually dimorphic patterns of behavior.