African Frog Research Articles

Some Characteristics of Reproduction in the Natural Environment of the African Giant Frog (Conraua goliath) (Boulenger, 1906) In the Monomodal Forest Zone of Cameroon

Some Characteristics of Reproduction in the Natural Environment of the African Giant Frog (Conraua goliath) (Boulenger, 1906) In the Monomodal Forest Zone of Cameroon

Evolutionary relationships in the African frog family Ptychadenidae, including the first molecular analysis, range extension, and distribution modelling of the monotypic genus Lanzarana

ABSTRACT The family Ptychadenidae contains three ecologically and morphologically distinct genera: the widespread, species-rich and above-ground dwelling Ptychadena, the less widespread and diverse, burrowing Hildebrandtia, and the little-known, burrowing, monotypic Lanzarana, endemic to Somalia. Previous morphological studies placed Lanzarana in a sister position to Hildebrandtia and these two genera as sister to Ptychadena. However, our analyses of mitochondrial and multilocus nuclear DNA data revealed Lanzarana as a sister lineage to Ptychadena (divergence ∼28 Mya), and Hildebrandtia sister to them (∼42 Mya). This suggests that the common ancestor of ptychadenids may have exhibited a burrowing lifestyle similar to Hildebrandtia and Lanzarana, rather than the more active, above-ground lifestyle of Ptychadena. In addition, based upon analysis of available mitochondrial DNA data, we have identified major clades within the genus Ptychadena and propose them as four species groups, with the P. mascareniensis group being the first diverging lineage (∼20 Mya). We also report four new localities of Lanzarana largeni from central Somaliland, south-western Puntland, southern Somalia and south-eastern Ethiopia, extending the presently known range of the species westward. This represents the first official record of L. largeni from Ethiopia. An ecological niche model of L. largeni for present climatic conditions identifies extensive climatically highly suitable areas in central Somalia and south-eastern Ethiopia. A model for future climatic conditions estimates a substantial extension of climatically suitable areas to northern Somalia and north-eastern Kenya, likely due to predicted more humid conditions accompanying the increase of temperatures in the western Indian Ocean.

Research progress of neurotrophic factor 4 in nerve injury repair

Neurotrophin-4 (NT-4), the fourth member of the nerve growth factor family (NGFs), was found in vipers and African toad in 1991, and it was named for its similar gene sequence structure and biological activity with the first three growth factors of NGFs. Up to now, much evidence has addressed the role of NT-4 in nerve injury and repair. With the study of NT-4 gradually deepening, a certain understanding of its biochemical characteristics and distribution in tissues needs to be clarified. This paper gives a brief review of the biological role of NT-4 and its relationship with nerve injury repair. It gives a preliminary discussion on the role of NT-4 in nerve injury repair.

Environmental variation predicts patterns of genomic variation in an African tropical forest frog

Central African rainforests are predicted to be disproportionately affected by future climate change. How species will cope with these changes is unclear, but rapid environmental changes will likely impose strong selection pressures. Here we examined environmental drivers of genomic variation in the central African puddle frog (Phrynobatrachus auritus) to identify areas of elevated environmentally-associated turnover. We also compared current and future climate models to pinpoint areas of high genomic vulnerability where allele frequencies will have to shift the most in order to keep pace with future climate change. Neither physical landscape barriers nor the effects of past Pleistocene refugia influenced genomic differentiation. Alternatively, geographic distance and seasonal aspects of precipitation are the most important drivers of SNP allele frequency variation. Patterns of genomic differentiation coincided with key ecological gradients across the forest-savanna ecotone, montane areas, and a coastal to interior rainfall gradient. Areas of greatest vulnerability were found in the lower Sanaga basin, the southeastern region of Cameroon, and southwest Gabon. In contrast with past conservation efforts that have focused on hotspots of species richness or endemism, our findings highlight the importance of maintaining environmentally heterogeneous landscapes to preserve genomic variation and ongoing evolutionary processes in the face of climate change.

Expanding the Genetic Code of Xenopus laevis Embryos.

The incorporation of unnatural amino acids into proteins through genetic code expansion has been successfully adapted to African claw-toed frog embryos. Six unique unnatural amino acids are incorporated site-specifically into proteins and demonstrate robust and reliable protein expression. Of these amino acids, several are caged analogues that can be used to establish conditional control over enzymatic activity. Using light or small molecule triggers, we exhibit activation and tunability of protein functions in live embryos. This approach was then applied to optical control over the activity of a RASopathy mutant of NRAS, taking advantage of generating explant cultures from Xenopus. Taken together, genetic code expansion is a robust approach in the Xenopus model to incorporate novel chemical functionalities into proteins of interest to study their function and role in a complex biological setting.

Species- and sex-specific chemical composition from an internal gland-like tissue of an African frog family.

Intraspecific chemical communication in frogs is understudied and the few published cases are limited to externally visible and male-specific breeding glands. Frogs of the family Odontobatrachidae, a West African endemic complex of five morphologically cryptic species, have large, fatty gland-like strands along their lower mandible. We investigated the general anatomy of this gland-like strand and analysed its chemical composition. We found the strand to be present in males and females of all species. The strand varies in markedness, with well-developed strands usually found in reproductively active individuals. The strands are situated under particularly thin skin sections, the vocal sac in male frogs and a respective area in females. Gas-chromatography/mass spectrometry and multivariate analysis revealed that the strands contain sex- and species-specific chemical profiles, which are consistent across geographically distant populations. The profiles varied between reproductive and non-reproductive individuals. These results indicate that the mandibular strands in the Odontobatrachidae comprise a so far overlooked structure (potentially a gland) that most likely plays a role in the mating and/or breeding behaviour of the five Odontobatrachus species. Our results highlight the relevance of multimodal signalling in anurans, and indicate that chemical communication in frogs may not be restricted to sexually dimorphic, apparent skin glands.

A double ovulation protocol for Xenopus laevis produces doubled fertilisation yield and moderately transiently elevated corticosterone levels without loss of egg quality.

The African claw-toed frog, Xenopus laevis, is a well-established laboratory model for the biology of vertebrate oogenesis, fertilisation, and development at embryonic, larval, and metamorphic stages. For ovulation, X. laevis females are usually injected with chorionic gonadotropin, whereupon they lay typically hundreds to thousands of eggs in a day. After being rested for a minimum of three months, animals are re-used. The literature suggests that adult females can lay much larger numbers of eggs in a short period. Here, we compared the standard "single ovulation" protocol with a "double ovulation" protocol, in which females were ovulated, then re-ovulated after seven days and then rested for three months before re-use. We quantified egg number, fertilisation rate (development to cleavage stage), and corticosterone secretion rate as a measure of stress response for the two protocol groups over seven 3-month cycles. We found no differences in egg number-per-ovulation or egg quality between the groups and no long-term changes in any measures over the 21-month trial period. Corticosterone secretion was elevated by ovulation, similarly for the single ovulation as for the first ovulation in the double-ovulation protocol, but more highly for the second ovulation (to a level comparable to that seen following shipment) in the latter. However, both groups exhibited the same baseline secretion rates by the time of the subsequent cycle. Double ovulation is thus transiently more stressful/demanding than single ovulation but within the levels routinely experienced by laboratory X. laevis. Noting that "stress hormone" corticosterone/cortisol secretion is linked to physiological processes, such as ovulation, that are not necessarily harmful to the individual, we suggest that the benefits of a doubling in egg yield-per-cycle per animal without loss of egg quality or signs of acute or long-term harm may outweigh the relatively modest and transient corticosterone elevation we observed. The double ovulation protocol therefore represents a potential new standard practice for promoting the "3Rs" (animal use reduction, refinement and replacement) mission for Xenopus research.

Sequences, phylogeny and evolution of mitochondrial delta-1-pyrroline-5-carboxylate dehydrogenases (ALDH4A1). Evidence for a second locus (ALDH4A2) in Drosophila

ALDH4A1 genes encode mitochondrial enzymes of delta-1-pyrroline-5-carboxylate metabolism, generating glutamate from either proline or ornithine. Analyses were undertaken of several vertebrate and invertebrate genomes using reported human and mouse ALDH4A1 amino acid sequences. ALDH4A1 sequences and structures were highly conserved, including residues involved in catalysis, coenzyme binding and enzyme structure, previously reported for mouse and human ALDH4A1. The human ALDH4A1 gene contained 15 coding exons and was more highly expressed in human liver and kidney cortex. Vertebrate ALDH4A1 mitochondrial leader sequences exhibited diverse sequences. Phylogeny studies supported the appearance of the ALDH4A1 gene in invertebrate evolution which has been conserved and retained throughout subsequent vertebrate evolution as a single ALDH4A1 gene. Exceptions included polyploidy observed for the Atlantic salmon (Salmo salar) and African toad (Xenopus laevis) genes. An examination of ALDH4A1 sequences from related Drosophila species supported the appearance of a second ALDH4A gene (ALDH4A2) and time dependent evolutionary changes over the past 50 million years for both genes.

Taxonomic re-evaluation of African anuran trypanosomes with the redescription and molecular diagnosis of Trypanosoma (Trypanosoma) nelspruitense Laveran, 1904 and Trypanosoma (Haematomonas) grandicolor Pienaar, 1962.

The aquatic and terrestrial clades of species of Trypanosoma could provide insight into the evolutionary history of the genus, as well as complementary information for biomedical studies of medically and economically important species of Trypanosoma. The ecological interactions and phylogeny of aquatic trypanosomes are currently not well-understood, mostly due to their complex life cycles and a deficiency of data. The species of Trypanosoma from African anuran hosts are of the least understood taxa in the genus. Trypanosomes were collected from South African frogs and subjected to morphological and phylogenetic analyses. This study redescribes Trypanosoma (Trypanosoma) nelspruitense Laveran, 1904 and Trypanosoma (Haematomonas) grandicolor Pienaar, 1962, with morphological and molecular data. The present study aims to create a platform for further future research on African anuran trypanosomes.

Septicemia caused by an emerging pathogen, Elizabethkingia miricola, in a laboratory colony of African dwarf frogs (Hymenochirus curtipes).

An outbreak of morbidity and mortality in an African dwarf frog (Hymenochirus curtipes) colony was reported following arrival at an animal research facility. Animals were found dead on arrival or became moribund shortly thereafter, and additional animals showed clinical signs of lethargy, weight loss, and anorexia over the following 3 weeks. Externally, some affected animals presented with multifocal areas of hyperemia in the inguinal and axillary areas and on the limbs, and mottled tan discoloration along the ventral abdomen. Histologically, lesions were consistent with generalized septicemia, characterized by granulomatous meningitis, otitis media, peritonitis (coelomitis), myocarditis and pericarditis, nephritis, pneumonia, and arthritis. Gram staining identified gram-negative rod-shaped bacteria free within tissues and within macrophages. Culture results of coelomic swabs identified moderate to numerous Elizabethkingia miricola. Testing of water from tanks housing affected animals showed elevated levels of nitrites and ammonia, and the presence of Citrobacter, Aeromonas, Pseudomonas, and Staphylococcus spp. cultured from several tank biofilters. E miricola is a newly recognized and rapidly emerging opportunistic pathogen in anurans and has been reported as a cause of septicemia in humans. This report documents the first occurrence of E. miricola septicemia in African dwarf frogs and illustrates the importance of this potential pathogen in the laboratory setting for amphibian research colonies, as well as those individuals directly working with them.

THE ROLE OF ADRENERGIC RECEPTORS IN THE ACTION OF ADRENALINE ON PLASMA LACTATE, GLUCOSE, LIVER, AND SKELETAL MUSCLE GLYCOGEN IN THE COMMON AFRICAN TOAD BUFO REGULARIS

In mammals, the roles of liver and skeletal muscle glycogen in adrenaline-induced hyperglycaemia are well known. However, in amphibians, there is limited information on the relative contribution of liver and skeletal muscle glycogen in adrenaline-induced hyperglycaemia. This study investigated the effects of adrenaline on blood glucose, plasma lactate, liverand skeletal muscle glycogen and the role of adrenergic receptors in the common African toad, bufo regularis. One hundred and twenty-five adult common African toads (70-100g) of both sexes were randomly collected and used in the study.Blood samples were collected from truncus arteriosus to estimate glucose and lactate levels. Blood glucose was determined immediately by modified glucose oxidase method. Plasma lactate was determined by modification of the Barker-Summerson method. Liver and gastrocnemius muscle glycogen were determined using anthrone reagents method. Adrenaline caused significant increase in blood glucose, lactate levels and significant reduction in liver and muscle glycogen. When toads were pre-treated with propranolol or prazosin, adrenalines reduction in liver and muscle glycogen was significantly reduced while the increase in blood glucose and lactate levels was prevented. Combination of both blockers abolished the increase in blood glucose, lactate levels and blocked reduction in liver and muscle glycogen produced by adrenaline. The results of this study showed that adrenalinecaused liver and skeletal muscle glycogen breakdown, increase in plasma lactate levels which resulted in hyperglycaemia in the common African toad bufo regularis. The study also showed that both alpha- and beta-adrenergic receptors are involved in mediating the effects of adrenaline on blood glucose, plasma lactate, liver, and muscle glycogenolysis and gluconeogenesis.

Молекулярное моделирование конформационной подвижности молекулы гиламбатина

The features of the spatial organization of the hylambatin molecule were investigated by methods of molecular mechanics and molecular dynamics. Hylambatin consists of twelve amino acid residues in the sequence: Asp-Pro-Pro-Asp-Pro-Asn-Arg-Phe-Tyr-Gly-Met-Met-NH2. Unlike all other tachykinins, hylambatin has a Met residue replacing the usual Leu at penultimate position. The tachykinin peptide hylambatin has been isolated and chemically characterized from methanol extracts of the skin of Hylambates maculatus, an African rhacophorid frog. It has been shown that intravenously administered hylambatin significantly increases the level of glucose and insulin in blood plasma. In this paper, the conformational flexibility of the hylambatin molecule was studied by methods of molecular mechanics and molecular dynamics. The conformational calculation of the peptide took into account non-valent and electrostatic interactions, hydrogen bonds and torsion potentials. Based on fragmentary analysis, stable spatial structures of the hylambatin dodecapeptide were determined, which can be represented as a set of conformations characterized by a relatively labile N-terminal tetrapeptide and a conformationally rigid C-terminal octapeptide. In the calculated stable conformational states, the effective interactions of the side chains of residues and hydrogen bonds were refined and energetically evaluated. It has been shown that the hylambatin molecule preferably forms practically isoenergetic conformations with various structural types at the N-end of the peptide chain, passing into the alpha helix at the C-end. By the method of molecular dynamics, the pattern of intramolecular mobility of stable conformations of the hylambatin molecule was modeled both in vacuum and surrounded by water molecules. Based on the calculated values of the dihedral angles, molecular models of energetically preferred conformational states of the hylambatin dodecapeptide were constructed.

Genomic Analyses Reveal Association of ASIP with a Recurrently evolving Adaptive Color Pattern in Frogs.

Traits shared among distantly related lineages are indicators of common evolutionary constraints, at the ecological, physiological, or molecular level. Here, we show that the vertebral stripe, a cryptic color pattern, has evolved hundreds of times in the evolutionary history of anurans (frogs and toads) and is favored in terrestrial habitats. Using a genome-wide association study, we demonstrate that variation near the Agouti signaling protein gene (ASIP) is responsible for the different vertebral stripe phenotypes in the African grass frog Ptychadena robeensis. RNAseq and real-time quantitative PCR revealed that differential expression of the gene and an adjacent long non-coding RNA is linked to patterning in this species. Surprisingly, and although the stripe phenotypes are shared with closely related species, we found that the P. robeensis alleles are private to the species and unlikely to evolve under long-term balancing selection, thus indicating that the vertebral stripe phenotypes result from parallel evolution within the group. Our findings demonstrate that this cryptic color pattern evolved rapidly and recurrently in terrestrial anurans, and therefore constitutes an ideal system to study repeated evolution.

More time for aliens? Performance shifts lead to increased activity time budgets propelling invasion success

In the Grinnellian niche concept, the realized niche and potential distribution is characterized as an interplay among the fundamental niche, biotic interactions and geographic accessibility. Climate is one of the main drivers for this concept and is essential to predict a taxon’s distribution. Mechanistic approaches can be useful tools, which use fitness-related aspects like locomotor performance and critical thermal limits to predict the potential distribution of an organism. These mechanistic approaches allow the inclusion key ecological processes like local adaptation and can account for thermal performance traits of different life-history stages. The African Clawed Frog, Xenopus laevis, is a highly invasive species occurring on five continents. The French population is of special interest due to an ongoing expansion for 40 years and a broad base of knowledge. We hypothesize that (1) the French population exhibits increased activity time in the invasive European range that could be devoted to fitness-relevant activity and (2) tadpoles may have less activity time available than adult frogs from the same range. We investigate how thermal performance traits translate into activity time budgets and how local adaptation and differences in the thermal responses of life-history stages may boost the European Xenopus invasion. We use a mechanistic approach based on generalized additive mixed models, where thermal performance curves were used to predict the hours of activity and to compare the potential activity time budgets for two life-history stages of native and invasive populations. Our results show that adult French frogs have more activity time available in Europe compared to South African frogs, which might be an advantage in searching for prey or escaping from predators. However, French tadpoles do not have more activity time in Europe compared to the native South African populations suggesting that tadpoles do not suffer the same strong selective pressure as adult frogs.

Diet of The North African Green Frog Pelophylax saharicus in The Wetlands of Edough (north- eastern of Algeria)

Diet of The North African Green Frog Pelophylax saharicus in The Wetlands of Edough (north- eastern of Algeria)

Trends to adaptation of the Sahara frog (Pelophylax saharicus) larvae across an environmental gradient

Species adjust their behavior and life-history to adapt to local environmental conditions. Species with a broad ecological niche often show signatures of local adaptations to different environment, particularly in extreme ones. Here, we investigate local adaptation in different populations of the North African Sahara frog (Pelophylax saharicus) living in various environmental conditions that vary mostly in temperature, precipitation, and elevation by mean of common garden experiment aiming to estimate the growth rate under two predation treatments (absence or presence of non-lethal cues of dragonfly larvae). First, we found an elevational cline in the reproductive phenology where, from low to high elevation, the reproductive season shifts to later dates, whereas that in arid environment was later than all other populations. We suggest that geographic differences in temperature and rainfall (in arid areas) explain this phenological pattern. Second, hatching success was overall high but showed a slight decline across elevation. Third, growth rate was generally faster in low and intermediate elevation populations, but slower in high elevation and arid environment populations. Populations in low and intermediate elevation responded to predation by reducing growth rate and the size at metamorphosis, but no predatory responses were recorded in high elevation and arid environment populations. Our study shows some life history signatures of local adaptation of P. saharicus in Northeast Algeria, which does not go in line with recent genetic analysis showing low population differentiation in the region.

Comparative study of fasting blood glucose and haematological parameters of Wistar rat, Rattus norvegicus and the common African toad, Bufo regularis

Haematological parameters are good indicators of the physiological status of animals and useful in understanding the relationship of blood to the environment. This study was carried out to compare the fasting levels of blood glucose and haematological parameters (Red blood cell count, Packed cell volume, Haemoglobin concentration, White blood cell count, Platelet count, and differential White blood cell counts) in Wistar rats, Rattus norvegicus and the common African toad, Bufo regularis. Ten rats and ten toads were used in the study. The animals were divided into two groups. Group I consists of 10 rats while Group II consists of 10 toads. The animals were fasted overnight before blood samples were collected for the determination of blood glucose and blood parameters. The blood glucose was determined using modified glucose oxidase method while the blood parameters were determined using standard laboratory techniques. The results of the study showed that the fasting blood glucose level in rats was significantly higher than that of toad. The fasting levels of blood parameters, packed cell volume, red blood cell counts, haemoglobin concentration in the toad were significantly lower than those of rats while the white blood cell counts of the toads were significantly higher compared with the rats. There were no significant differences in neutrophil, monocyte, eosinophil and lymphocyte counts of rats when compared with those of the toad.

Systematic position of the Clicking Frog (Kassinula Laurent, 1940), the problem of chimeric sequences and the revised classification of the family Hyperoliidae

The systematics of the African frog family Hyperoliidae has undergone turbulent changes in last decades. Representatives of several genera have not been genetically investigated or with only limited data, and their phylogenetic positions are thus still not reliably known. This is the case of the De Witte's Clicking Frog (Kassinula wittei) which belongs to a monotypic genus. This miniature frog occurs in a poorly studied region, southeastern Democratic Republic of the Congo, northern Zambia, Angola. So far it is not settled whether this genus belongs to the subfamily Kassininae as a relative of the genus Kassina, or to the subfamily Hyperoliinae as a relative of the genus Afrixalus. Here we present for the first time a multilocus phylogenetic reconstruction (using five nuclear and one mitochondrial marker) of the family Hyperoliidae, including Kassinula. We demonstrate with high confidence that Kassinula is a member of Hyperoliinae belonging to a clade also containing Afrixalus (sub-Saharan Africa), Heterixalus (Madagascar) and Tachycnemis (Seychelles). We find that Kassinula represents a divergent lineage (17–25 Mya), which supports its separate genus-level status, but its exact systematic position remains uncertain. We propose to name the clade to which the above four genera belong as the tribe Tachycnemini Channing, 1989. A new taxonomy of the family Hyperoliidae was recently proposed by Dubois et al. (2021: Megataxa 5, 1–738). We demonstrate here that the new taxonomy was based on a partially erroneous phylogenetic reconstruction resulting from a supermatrix analysis of chimeric DNA sequences combining data from two families, Hyperoliidae and Arthroleptidae (the case of Cryptothylax). We therefore correct the erroneous part and propose a new, revised suprageneric taxonomy of the family Hyperoliidae. We also emphasize the importance of inspecting individual genetic markers before their concatenation or coalescent-based tree reconstructions to avoid analyses of chimeric DNA sequences producing incorrect phylogenetic reconstructions. Especially when phylogenetic reconstructions are used to propose taxonomies and systematic classifications.

Phylogeny and taxonomy of the African frog genus Strongylopus (Anura: Pyxicephalidae)

We present a molecular phylogeny of African stream frogs (genus Strongylopus), based on 12S rRNA, 16S rRNA, the nuclear recombination activating gene 1 (RAG-1) and tyrosinase exon 1 (tyr). Molecular data were supported by advertisement call analysis and morphology. We recognise six valid species: Strongylopus bonaespei (Dubois, 1981) from the southern and southwestern parts of the Western Cape Province, South Africa; Strongylopus fasciatus (Smith, 1849) from eastern South Africa to Zimbabwe; Strongylopus grayii (Smith, 1849) found throughout South Africa with older records in Naukluft, in central Namibia; Strongylopus rhodesianus (Hewitt, 1933) known from the eastern highlands of Zimbabwe and western Mozambique; Strongylopus wageri (Wager, 1961) from KwaZulu-Natal Province, South Africa and Strongylopus merumontanus (Lönnberg, 1907) from eastern Zambia, Malawi, northern Mozambique and Tanzania. Strongylopus fuelleborni (Nieden, 1911), S. kitumbeine Channing & Davenport, 2002 and S. kilimanjaro Clarke & Poynton, 2005 were shown to be junior synonyms of Strongylopus merumontanus. Strongylopus springbokensis Channing, 1986 is recovered as a junior synonym of Strongylopus grayii. Divergence ages were estimated, and discussed in terms of paleoclimatic events.

Characterization of Some Novel Antimicrobial Peptides from African Common Toad, Sclerophrys regularis

The growing number of antimicrobial resistant bacteria have necessitated urgent need for newer and effective antimicrobials. Antimicrobial peptides (AMPs) which are also called host defense peptides are promising class of bioactives found in all living thing especially amphibians. A total of 355 Sclerophrys regularis were collected and each toad was weighed and 0.02mL/g of epinephrine (1mg/mL) was administered via the dorsal lymph sac of the toad. The skin secretions were washed, lyophilized and stored at -20°C. The secretion was purified using Sephadex G-50 gel purification column. The fractions showing antimicrobial activities against Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 25922, Salmonella typhii ATCC 14028, Citrobacter freundii ATCC 8090, Staphylococcus aureus ATCC 25913 and Candida albicans ATCC 3147 were further purified by RP- HPFC. The purified fractions were lyophilized and m/z ratio peaks were determined using MALDI TOF/TOF MS. The sequences corresponding to the peaks were determined using Mascot Peptide Mass Fingerprint which revealed novel AMP sequences: IHAGKTVPIVK, MHLLWR and LTGQIKNGLSGR which were named Regularin-2, Regularin-3 and Regularin-4 respectively. The MICs of purified fractions were between 50 – 200 µg/mL and the antimicrobial activities were maintained when stored at -20 °C for 180 days and was significant at p < 0.05. Bioprospecting of Nigerian toad specie (S. regularis) could yield potential therapeutic AMP agents.