Larval bullfrog skin expresses ENaC despite having no amiloride-blockable transepithelial Na+ transport

Amiloride-blockable Na⁺ transport, measured as an amiloride-blockable short-circuit current (Am-SCC), is mediated by the epithelial Na⁺ channel (ENaC). Am-SCC is not normally present in bullfrog tadpole skin, but when such skin is cultured with corticoids an amiloride-blockable Na transport appears. Prolactin (PRL) inhibits its corticoid-induced development. Using specific PCR primers for adult frog ENaC and RT-PCR, we investigated whether corticoids can induce all three ENaC subunits, and whether this expression of ENaC subunit(s) can be blocked by adding PRL with the corticoids. We found that (1) the sequences of the RT-PCR products obtained using primers for α-ENaC were identical between larval and adult skins, (2) the mRNAs for all three ENaC subunits were expressed in larval skin under normal conditions despite no amiloride-blockable Na⁺ transport being detectable, (3) all three subunits were expressed in larval skins whether they were cultured with corticoids (amiloride-blockable Na transport present) or with corticoids supplemented with PRL (no amiloride-blockable Na transport present). An antibody against a peptide from the α-ENaC of adult bullfrog was localized to the apical cells of both larval and adult skins. Since no amiloride-blockable Na transport exists across larval skin under these conditions, these results suggest that ENaC protein was expressed prior to the onset of transport. ENaC may be in the plasma membrane in an inactivated form or, alternatively, within vesicles waiting to be inserted.     

Heme oxygenase activity in liver microsomes of the bullfrog, Rana catesbeiana, and its change during the tadpole development

Heme oxygenase in the liver microsomes of tadpole and adult bullfrog, Rana catesbeiana, was studied in relation to hemoglobin metabolism during tadpole development. The results obtained are as follows. 1. The specific activity of the enzyme of premetamorphic tadpole liver was comparable to that of adult frog liver. The apparent Km for methemalbumin was about 25 microM for both tadpole and frog enzymes. 2. The enzyme activity was stimulated in vivo by the injection of methemalbumin, phenylhydrazine and triiodothyronine to the animals. 3. A marked increase in the enzyme activity was found in the liver of tadpole during prometamorphic stage and metamorphic climax.     

Gut microbiota of invasive bullfrog tadpoles responds more rapidly to temperature than a noninvasive congener

Environmental temperature can alter the composition, diversity, and function of ectothermic vertebrate gut microbial communities, which may result in negative consequences for host physiology, or conversely, increase phenotypic plasticity and persistence in harsh conditions. The magnitude of either of these effects will depend on the length of time animals are exposed to extreme temperatures, and how quickly the composition and function of the gut microbiota can respond to temperature change. However, the temporal effects of temperature on gut microbiota are currently unknown. Here, we investigated the length of time required for increased temperature to alter the composition of gut bacterial communities in tadpoles of two frog species, the green frog, Lithobates clamitans, and its congener, the globally invasive American bullfrog, L. catesbeianus. We also explored the potential functional consequences of these changes by comparing predicted metagenomic profiles across temperature treatments at the last experimental time point. Bullfrog‐associated microbial communities were more plastic than those of the green frog. Specifically, bullfrog communities were altered by increased temperature within hours, while green frog communities took multiple days to exhibit significant changes. Further, over ten times more bullfrog bacterial functional pathways were temperature‐dependent compared to the green frog. These results support our hypothesis that bullfrog gut microbial communities would respond more rapidly to temperature change, potentially bolstering their ability to exploit novel environments. More broadly, we have revealed that even short‐term increases in environmental temperature, expected to occur frequently under global climate change, can alter the gut microbiota of ectothermic vertebrates.     

Helminth and leech community structure in tadpoles and caudatan larvae of two amphibian species from Western Nebraska

Currently no comparative studies exist on helminth and leech community structure among sympatric anuran tadpoles and salamander larvae. During June-August 2007-2009, we examined 50 bullfrog tadpoles, Rana catesbeiana , 50 barred tiger salamander larvae, Ambystoma mavortium , and 3 species of snails from Nevens Pond, Keith County, Nebraska for helminth and leech infections. The helminth and leech compound community of this larval amphibian assemblage consisted of at least 7 species, 4 in bullfrog tadpoles and 4 in barred tiger salamander larvae. Bullfrog tadpoles were infected with 2 species of nematodes ( Gyrinicola batrachiensis and Spiroxys sp.) and 2 types of metacercariae ( Telorchis sp. and echinostomatids), whereas barred tiger salamander larva were infected with 1 species of leech ( Placobdella picta ), 2 species of adult trematodes ( Telorchis corti and Halipegus sp.), and 1 species of an unidentified metacercaria. The component community of bullfrog tadpoles was dominated by helminths acquired through active penetration, or incidentally ingested through respiratory currents, or both, whereas the component community of larval salamanders was dominated by helminths acquired through ingestion of intermediate hosts (χ2 = 3,455.00, P < 0.00001). Differences in amphibian larval developmental time (2-3 yr for bullfrog tadpoles versus 2-5 mo for salamander larvae), the ephemeral nature of intermediate hosts in Nevens Pond, and the ability of bullfrog tadpole to eliminate echinostome infections had significant effects on mean helminth species richness among amphibian species and years (t = 12.31, P < 0.0001; t = 2.09, P = 0.04). Differences in herbivorous and carnivorous diet and time to metamorphosis among bullfrog tadpoles and barred tiger salamander larvae were important factors in structuring helminth communities among the larval stages of these 2 sympatric amphibian species, whereas size was important in structuring helminth and leech communities in larval salamanders, but not in bullfrog tadpoles.     

Reduced calcium and inhibition of protein kinase C mimic the enhancement of ornithine decarboxylase activity of prolactin in Ambystoma tigrinum tissues

We previously reported that prolactin (PRL) could increase the activity of ornithine decarboxylase (ODC) in liver slices taken from larval tiger salamanders (Ambystoma tigrinum). This action of the hormone was inhibited by oxytocin (OT), the calcium ionophore A23187, and diacyglycerol (DG) and was duplicated by 10 microM verapamil (VML), a calcium channel blocker. Here, we expand these results to show that 1) a higher dose of VML (50 microM) produces an additive effect with PRL; 2) addition of small amounts of calcium (0.1 mM) to the liver culture medium blocks PRL action; 3) neither nifedipine (NIF), a different type of calcium channel blocker, nor EDTA alter PRL action; and 4) gossypol, a reported inhibitor of protein kinase C, mimics PRL action. Additionally, we show that PRL increases ODC activity in tiger salamander tail skin in vitro, a tissue previously demonstrated to be a PRL target tissue in this species. The same set of treatments which we have shown to modify PRL effects on ODC in liver slices affects PRL action in the tail skin in a parallel manner. Thus, the mechanism whereby PRL enhances ODC activity appears to be the same in both these tissues. These results are discussed in conjunction with the findings from similar studies using mammalian tissues in an attempt to assess the current picture of the mechanism of PRL action and the possible role of inositol phospholipid turnover, calcium, and protein kinase C in the action of this hormone.     

Comparative study on vertebrate liver AMP deaminases

Similar activity of AMP deaminase was found in rat, hen, turtle and flounder liver when estimated at high AMP concentration. The enzyme activity was of an order of magnitude higher in frog liver. Simple step by step phosphocellulose column chromatography revealed two forms of AMP deaminase in chicken and flounder liver and one form in the liver of rat and turtle. All enzymes (except for frog liver AMP deaminase) were activated by ATP. The enzymes from rat, frog and both forms from flounder were also activated by ADP. GTP exhibited a variety of effects. The enzyme from rat and turtle was inhibited, both forms from hen and flounder were activated and frog liver enzyme was not influenced.     

Immunocytochemical demonstration of growth hormone,prolactin and somatostatin-like immunoreactivities in the brain of larval,young adult and upstream migrant adult sea lamprey,Petromyzon marinus

Summary Growth hormone, prolactin and somatostatinlike immunoreactivities were demonstrated in the brains of larval, young adult (parasitic) and upstream migrant adult sea lampreys, Petromyzon marinus, by means of immunoperoxidase techniques. Growth hormone (GH) and prolactin (PRL) were observed within separate perikarya in the nucleus praeopticus, within fibers in the commissura praeinfundibularis, and in nerve endings within the neurohypophysis of larval and adult-stage lampreys. Cell bodies demonstrating immunoreactive growth hormone were more numerous than those reactive for prolactin. Unlike in the upstream migrant adult lamprey, no GH or PRL was demonstrated in the adenohypophysis of larval or parasitic lamprey.Somatostatin (SRIF)-like immunoreactive neurons were demonstrated in the nucleus commissurae praeinfundibularis, anterior and posterior pars ventralis hypothalami, pars dorsalis thalami, and the tegmentum motorium rhombencephali of larval, parasitic and upstream migrant adult lampreys. Many of the SRIF containing neurons within the hypothalamus were cerebrospinal fluid (CSF)-contacting cells. SRIF fibers were found throughout most of the brain predominating within the nucleus praeopticus, pars ventralis hypothalami, and the nucleus interpeduncularis. No SRIF immunoreactivity was found within the neurophyophysis. The possible functions of these peptides within the brain of the lamprey are discussed.     

Identification of tilapia ghrelin and its effects on growth hormone and prolactin release in the tilapia,Oreochromis mossambicus

We have identified ghrelin and cDNA encoding precursor protein from the stomach of a euryhaline tilapia, Oreochromis mossambicus. The sequence of 20-amino acid tilapia ghrelin is GSSFLSPSQKPQNKVKSSRI. The third serine residue was modified by n-decanoic acid. The carboxyl-terminal end of the peptide possessed an amide structure. RT-PCR analysis revealed high levels of gene expression in the stomach and low levels in the brain, kidney and gill. Tilapia ghrelin stimulated growth hormone (GH) and prolactin (PRL) release from the organ-cultured tilapia pituitary at a dose of 10 nM. Thus, a novel regulatory mechanism of GH secretion by gastric ghrelin seems to be conserved in the tilapia. Stimulation of PRL release by homologous ghrelin has been reported in human, bullfrog and eel, and suggests the presence of growth hormone secretagogue receptor not only on somatotrophs but also on PRL cells of the tilapia pituitary.     

Acetylcholinesterase activity in the adrenal chromaffin vesicles of Urodela

The presence of acetylcholinesterase (AChE) activity in the adrenal chromaffin cells of Necturus maculosus and Ambystoma maculatum (Amphibia, Urodela) has been demonstrated by cytochemical method at the electron microscope level. The enzymatic activity is localized in RER and perinuclear cisternae, on the plasma membrane and within the chromaffin vesicles, both in adrenaline (A) and noradrenaline (N) cells. Moreover N cells appear to be more reactive than A cells and Necturus more reactive than Ambystoma. The possible function of the AChE activity inside the vesicles is discussed as a mechanism of protons donor or as peptidasic activity acting on various peptides present in the vesicle.     

The complete amino acid sequence of growth hormone of the bullfrog (Rana catesbeiana).

The primary structure of growth hormone (GH) isolated from the adenohypophysis of the bullfrogs (Rana catesbeiana) was determined. The hormone was reduced, carboxymethylated and subsequently cleaved with cyanogen bromide. Intact bullfrog GH was also digested with lysyl endopeptidase and trypsin. The resulting fragments were separated by reverse-phase high-performance liquid chromatography and subjected to sequence analysis using an automated gas-liquid sequencer employing the Edman method. Bullfrog GH was found to consist of 190 amino acid residues. The amino acid sequence determined is in accord with that deduced from bullfrog GH cDNA by Pan and Chang (1988) except for nine residues at positions 43-48, 73, 80 and 87. Sequence comparisons revealed that bullfrog GH is more similar to tetrapod GHs (e.g., 69% homology with sea turtle GH, 66% with chicken GH and 61% with ovine GH) than to GHs of teleosts (e.g., 35% homology with chum salmon GH and 33% with bonito GH) except for eel (52% identity). Bullfrog GH and prolactin exhibit a sequence homology of 25%.     

Molecular cloning and functional characterization of a prolactin-releasing peptide homolog from Xenopus laevis

Amino acid sequences for identified prolactin (PRL)-releasing peptides (PrRPs) were conserved in mammals (>90%) or teleost fishes (100%), but there were considerable differences between these classes in the sequence (<65%) as well as in the role of PrRP. In species other than fishes and mammals, we have identified frog PrRP. The cDNA encoding Xenopus laevis prepro-PrRP, which can generate putative PrRPs, was cloned and sequenced. Sequences for the coding region showed higher identity with teleost PrRPs than mammalian homologues, but suggested the occurrence of putative PrRPs of 20 and 31 residues as in mammals. The amino acid sequence of PrRP20 was only one residue different from teleost PrRP20, but shared 70% identity with mammalian PrRP20s. In primary cultures of bullfrog (Rana catesbeiana) pituitary cells, Xenopus PrRPs increased prolactin concentrations in culture medium to 130–160% of the control, but PrRPs was much less potent than thyrotropin-releasing hormone (TRH) causing a three- to four-fold increase in prolactin concentrations. PrRP mRNA levels in the developing Xenopus brain peak in early prometamorphosis, different from prolactin levels. PrRP may not be a major prolactin-releasing factor (PRF), at least in adult frogs, as in mammals.     

Calcium transport activities of plasma membranes isolated from the livers of various animal species

1. Plasma membranes of comparable yield and purity were isolated from the livers of various animal species belonging to phylogenetic groups from Amphibia to Mammalia. 2. Calcium transport activity was observed in all liver plasma membranes examined. 3. No phylogenetic pattern of expression of the liver plasma membrane calcium transport system was observed, with the order of activity being: guinea pig greater than rabbit greater than frog greater than chicken = hamster greater than rat = budgerigar = turtle greater than beef cattle greater than mouse = duck. 4. Calcium transport activity was only 9.7 and 8.7% of adult frog levels in plasma membranes isolated from the livers of tadpoles without and with limbs, respectively. 5. Liver plasma membrane calcium transport activity was 25% higher in adult chickens than in day-old chicks. 6. A possible role for thyroid hormone in the development of the liver plasma membrane calcium transport system is discussed.     

Complete amino acid sequences of a pair of fish (tilapia) prolactins, tPRL177 and tPRL188

The complete amino acid sequences of a pair of tilapia (Oreochromis mossambicus) prolactins (PRLs) were determined. The larger PRL of molecular mass 20,836 Da consists of 188 amino acid residues. The smaller PRL of molecular mass 19,584 Da is 11 residues shorter. On alignment of the two sequences, the 19.6-kDa PRL (tPRL177) has two conspicuous deletions on the NH2-terminal side of the disulfide bond which connects the first and second cysteine residues. The degree of similarity between the two PRL sequences is unexpectedly low (130 identical residues, 69%) compared with that between the variants of other teleostean PRLs. Circular dichroism spectra and hydropathy profiles suggest structural similarity of the two PRLs. The sequence of the 20.8-kDa PRL (tPRL188) has 69% identity with that of salmon PRL. The sequence of tPRL177 is 56% identical with that of salmon PRL. Each tilapia PRL is equally similar to mammalian PRLs (about 30% identical residues). Regions highly conserved among teleostean and mammalian PRLs were identified on the COOH-terminal side of the disulfide bond connecting the first and second cysteine residues.     

Serum prolactin levels and crop-sac development in ring doves during a breeding cycle

Using a turkey prolactin radioimmunoassay, the serum prolactin levels of male and female ring doves (Streptopelia risoria) during the breeding cycle were measured and their circulating prolactin levels were compared to crop-sac weight on a within-bird basis. During the early phase of the incubation period, crop weight showed a delayed response to prolactin stimulation and there was no correlation; during the midincubation period when prolactin and crop-sac weight were increasing, there was a strong positive correlation; around hatching, when prolactin was at its peak, there was no correlation. There were again strong positive correlations at later samples, when squabs were developing and both prolactin and crop-sac weight were declining. Thus, it appears that the correlation between the circulating prolactin level and crop-sac development depends on the stage of the breeding cycle. While males and females showed similar pattern of circulating prolactin during the period of incubation and parental care, only females consistently showed a postovulatory rise of prolactin. These results were discussed in the context of the role of prolactin in the breeding cycle.     

Cardiovascular responses of the bullfrog (Rana catesbeiana) to thermal stimulation of the spinal cord

Selective thermal stimulation of the spinal cord was performed in the bullfrog (Rana catesbeiana). Spinal cord warming caused an increase in systolic pressure of the truncus arteriosus, and cooling caused a decrease. Spinal cord warming caused an increase in systolic and pulse pressures of the conus arteriosus, and cooling caused a decrease. These results showed the temperature perceptibility of the spinal cord and a relationship between spinal cord temperature and autonomic functions in the frog as in other endothermic and ectothermic species.     

Homologous growth hormone (GH) binding in gilthead sea bream (Sparus aurata). Effect of fasting and refeeding on hepatic GH-binding and plasma somatomedin-like immunoreactivity

Specific binding of gilthead sea bream growth hormone (sbGH) to liver membrane preparations was a time and temperature dependent process, and was saturable by increasing amounts of membrane proteins. Scatchard analysis evidenced a single class of high-affinity and lowcapacity binding sites. Ovine prolactin, recombinant tilapia prolactin, carp gonadotropin and chinook salmon gonadotropin did not compete for the¹²⁵I-sbGH binding sites, while recombinant trout GH, bovine GH and human GH displaced iodinated sbGH in a dose dependent-manner. IGF-I-like immunoreactivity was detected after acidification of plasma and removal of IGF-I binding activity. A parallel displacement to the rhIGF-1 standard was observed with extracted plasma samples. Free and total hepatic GH-binding decreased during long-term starvation (3–9 weeks), returning to control values during the refeeding period. Plasma IGF-I-like immunoreactivity showed a similar trend. To our knowledge, this is the first report that indicates a coordinated regulation of GH-binding and plasma somatomedin-like activity in a typical marine fish.     

Larval skipper frogs recognise kairomones of certain predators innately

Recognising potential predators is critical for the survival and reproduction of prey animals. However, prey animals may possess an innate ability to recognise the signature odours (kairomones) of only certain native, sympatric predators, while requiring learning to recognise others. Our observations have shown that larval skipper frogs (Euphlyctis cyanophlyctis) fail to recognise kairomones of dragonfly nymph, a common predator of amphibian tadpoles with a cosmopolitan distribution. Hence, we wanted to determine if larval skipper frogs totally lack an innate mechanism to recognise kairomones of all aquatic predators, or have an innate ability to recognise kairomones of only certain predators. In a series of experiments, we tested the antipredator response of larval skipper frogs to kairomones of dragonfly nymph (Bradinopyga geminata); walking catfish (Clarias batrachus); Mozambique tilapia (Oreochromis mossambicus); two species of predatory tadpoles, Indian bullfrog (Hoplobatrachus tigerinus) and Jerdon’s bullfrog (Hoplobatrachus crassus); and the checkered keel back snake (Xenochrophis piscator). The results clearly indicate that larval skipper frogs have the innate ability to recognise kairomones of the walking catfish, both species of larval bullfrog and checkered keel back snake. However, they lack the innate ability to recognise kairomones of dragonfly nymph and Mozambique tilapia. Prey choice of the Mozambique tilapia and gape-limitation of dragonfly nymphs could be responsible for the lack of innate responses of larval skipper frogs to them. The study provides empirical evidence for the notion that prey can innately recognise certain predators.     

Thyroid gland development in a neotenic goby (ice goby,Leucopsarion petersii) and a common goby (ukigori,Gymnogobius urotaenia) during early life stages

In order to study the characteristics of neoteny in teleosts, development of the thyroid system and digestive tract of a neotenic goby (ice goby, Leucopsarion petersii) and a non-neotenic goby (ukigori, Gymnogobius urotaenia) were compared. In juvenile ukigori, the intestine was found to be convoluted once in the antero-midpart, and gastric glands were present. In the ice goby, the alimentary canal was straight, and no gastric gland was observed even in adult, suggesting that the ice goby retains larval features, not only in appearance but also in internal organs. A marked difference was also found in the thyroid system. In ukigori, activity of the thyroid gland and thyroid stimulating hormone (TSH) cells increased between flexion and postflexion larval phases. However, in the ice goby, thyroid glands remained inactive, and no TSH cells were observed. A delayed development of the thyroid system was suggested as a major factor contributing to neoteny in the ice goby.     

ERYTHROPOIESIS DURING AMPHIBIAN METAMORPHOSIS : II. Immunochemical Study of Larval and Adult Hemoglobins of Rana catesbeiana

Rabbit antibodies were prepared against the major hemoglobin components of the larval and adult stages of R. catesbeiana. The properties of the antisera were studied by double immunodiffusion, precipitation, and complement fixation. The antisera to tadpole and frog hemoglobins did not cross-react with either hemoglobin or apohemoglobin. The anti-serum against frog hemoglobin was used for the detection of frog hemoglobin in tadpoles undergoing either natural or thyroxine-induced metamorphosis. It was shown that frog hemoglobin is detectable first in the liver, indicating that the liver is the site of erythrocyte maturation during metamorphosis.