Male Frogs Research Articles

More than meets the eye: decrypting diversity reveals hidden interaction specificity between frogs and frog‐biting midges

1. Female frog‐biting midges (Diptera: Corethrellidae) eavesdrop on the nocturnal mating calls of their blood hosts – male frogs. Available data suggest variable degrees of specialisation among Corethrella‐host associations, with limited information on the mechanisms involved in host selection and partitioning on a community level.2. Our study provides a first comprehensive analysis of host interactions for a neotropical community of frog‐biting midges, based on both morphological and molecular genetic species delimitation. We used quantitative bipartite interaction networks to investigate host specificity among the midge‐frog community of La Gamba, Pacific lowland Costa Rica.3. Midges that were collected directly from frog hosts (16 frog species) showed more pronounced levels of specificity (network‐wide degree of specialisation: H2′ = 0.3) than those caught with acoustic traps broadcasting their calls (12 frog species; H2′ = 0.08). This indicates that, despite a rather generalist acoustic foraging behaviour, frog‐biting midges discriminate between potential hosts by using additional close‐range recognition cues.4. Based on COI and ITS2 sequencing data, we identified considerable levels of cryptic diversity within our five Corethrella morphotypes, with at least 17 distinct MOTUs of Corethrella in La Gamba. Including these MOTUs in bipartite network analyses produced higher resolution in species interactions, and increased estimators of network specificity (H2′ = 0.42).

Getting ready for a long bath: skin permeability decreases prior to aquatic breeding in male toads.

Vertebrate skin regulates exchanges between the organism and its environment and notably plays a fundamental role in regulating water fluxes. Dynamic changes of skin resistance to water fluxes are expected to occur in species that regularly shift between habitat types especially if these habitats differ in their hydric properties (e.g., terrestrial versus aquatic). We investigated changes of skin permeability using a study system (terrestrial toads) where reproduction induces a transition from terrestrial to freshwater habitats and a prolonged immersion that can last several weeks in males. In this system, the simultaneity between skin shedding and the onset of breeding suggests that the production of new integument layers prior to immersion for reproduction may regulate water influxes. We found that the skin permeability of male toads decreases significantly prior to breeding, suggesting that skin shedding at the onset of breeding regulates water fluxes to alleviate osmotic costs of immersion during reproduction. The continued decrease of skin permeability detected during breeding suggests that additional mechanisms interact with skin structure to further decrease permeability to water during a prolonged immersion. Future studies are required to assess whether changes in skin permeability to water tradeoffs with other skin characteristics (gas exchanges) relevant to aquatic breeding amphibians.

Male common midwife toad Alytes obstetricans depositing eggs in a flowerpot saucer in a suburban garden?

Male common midwife toad Alytes obstetricans depositing eggs in a flowerpot saucer in a suburban garden?

Linking in‐situ and ex‐situ populations of threatened amphibians through genome banking

AbstractGenetic variation of the endangered Puerto Rican crested toad (PRCT; Peltophryne lemur) has dwindled over time in both wild and captive populations, leading to long‐term sustainability issues for the recovery program. To address this challenge, we propose that PRCTs can be used as a model species to show how in‐situ and ex‐situ populations can be linked through sperm biobanking and gamete transfer, expanding genetic variability of both populations. Male toads (n = 10) in Guayanilla, Puerto Rico were administered human chorionic‐gonadotropin (hCG) and luteinizing‐hormone‐releasing‐hormone analog (GnRHa) to stimulate spermiation. Sperm was collected noninvasively from 9/10 males, cryopreserved in 10% trehalose with either 10% N,N‐dimethylformamide (DMFA) or 10% dimethyl sulfoxide (DMSO), and transported in liquid nitrogen vapor to the National Amphibian Genome Resource Bank at Mississippi State University. Ultrasonography was used to identify females (n = 3) with mature oocytes for GnRHa‐induced oviposition of eggs for in‐vitro fertilization (IVF). Post‐thaw sperm motility was 28% and 25% for sperm cryopreserved with DMFA or DMSO, respectively. Of the 9,672 eggs used for IVF, 4% (n = 306/6,981) were fertilized with frozen–thawed sperm, compared with 20% (n = 525/2691) fertilized with fresh sperm controls. Overall, 46 toadlets were produced from frozen–thawed sperm. After 4.5 months of headstarting, 14 juvenile toads produced from various genetic crosses using frozen–thawed sperm were released to new sites in the wild, introducing unique genetic representation and new founder lines. After 1.5 years, 24 adult toads produced using frozen–thawed sperm continue to thrive in the captive collection, and one of these males has now produced an F2 generation of offspring with 5,085 tadpoles released to the wild. This transformational study is the first to produce reproductively viable adult PRCTs using cryopreserved sperm from hormonally‐induced wild males and captive females, introducing innovative methods that link in‐situ and ex‐situ populations of endangered amphibians to revolutionize genetic management.

The cost of breeding for male common toads

Abstract The potential impact of pairing on male survivorship and future breeding success in common toads was investigated between 1993 and 2014. A total of 8132 males arriving at a breeding pond in southern England were measured, weighed, individually marked and their breeding history recorded. The body condition (BC) of all males was estimated using (a) residuals from an ordinary least squares regression analysis of log10 body mass against log10 body snout-vent length (SVL; ordinary least squares) and (b) a scaled mass index (SMI), standardized for SVL. Each year each male was allocated to one of four categorical groups: (1) Paired, (2) Unpaired, and whether or not it returned to breed the following year: (3) Return, (4) No Return. The BC of both paired and unpaired males that subsequently returned was significantly higher than that of those that failed to return. Male pairing success was highest in the youngest males and lowest in the oldest. The overall cost for male common toads that successfully paired was a significantly decreased BC and survival rate. First time breeding males had a 21.2% chance of surviving and returning to breed the following year, increasing to 28.3–37.0% for those returning for up to five consecutive years.

Influence of octylphenol on gene expression of gonadotropins and their receptors, testicular structure and mating behavior of male Rana chensinensis

In the present study, responses of the Chinese brown frog (Rana chensinensis) to exposure to different doses and duration of Octyphenol (OP) which degraded from alkylphenol ethoxylates (APEs) were characterized during the adult periods. The effects of OP on growth, development and reproduction and the expression of genes in gonad were investigated. The expression levels of fshβ, lhβ, fshr and lhr had significant differences as the exposure time increased. The pathological and morphological changes were also observed in the OP treatments. Furthermore, the number of TUNEL positive cells and the TUNEL index was elevated after exposed to OP. Besides that, OP treatment could influence its mating behavior and reduce the fertilization rates. Taken together, these results indicated that OP disrupt sex steroid signaling, normal development of spermatogenesis, courtship behavior of male frogs and decline fertilization rate in R. chensinensis.

Tissue-Specific Distribution of Legacy and Emerging Organophosphorus Flame Retardants and Plasticizers in Frogs.

Five types of tissues, including the liver, kidney, intestine, lung, and heart, were collected from black-spotted frogs and bullfrogs to study the tissue-specific accumulation of organophosphorus flame retardants (PFRs) and plasticizers. Thirteen PFRs and nine plasticizers were detected, with average total concentrations of 1.4–13 ng/g ww and 858–5503 ng/g ww in black-spotted frogs, 3.6–46 ng/g ww and 355–3504 ng/g ww in bullfrogs. Significant differences in pollutant concentrations among different tissues in the two frog species were found, indicating the specific selectivity distribution of PFRs and plasticizers. Overall, liver tissues exhibited significantly higher pollutant concentrations. The pollutant concentration ratios of other tissue to the sum of liver tissue and other tissues (OLR, Cother/(Cother + Cliver)) corresponding to male frogs were significantly greater than those of females, suggesting that male frogs could have higher metabolic potentials for PFRs and plasticizers. No obvious correlations between OLR and log KOW were found, indicating that the other factors (e.g., bioaccumulation pathway and metabolism) besides lipophicity could influence the deposition of PFRs and plasticizers in frog livers. Different parental transfer patterns for PFRs and plasticizers were observed in frogs when using different tissues as parental tissues. Moreover, the liver tissues had similar parental transfer mechanism with muscle tissues.

Assessing acoustic competition between sibling frog species using rhythm analysis.

Male frog advertisement calls are species‐specific vocalizations used to attract females for breeding. However, it is possible for environmental or biological sounds to overlap these calls in both frequency and duration resulting in signal confusion, influencing female decision and/or location abilities. It is therefore important for vocal species competing for the same acoustic space to partition their calls either spatially or temporally (via call alternation or suppression). However, frog species previously isolated from each other may not have developed appropriate adaptive behaviors, resulting in acoustic competition. This study applied rhythm analysis to track changes in calling behavior, namely changes in calling frequency (as in beats per second), of the wallum sedgefrog and the eastern sedgefrog when vocalizing alone versus in the presence of each other to assess potential acoustic competition. Our main findings demonstrated that both species significantly altered their calling behavior when exposed to each other. While we expected the increased calling activity of one species to inhibit the activity of the other to avoid signal confusion, we instead found that both species greatly increased the beat frequency of their calls when calling in the presence of each other. We also found evidence of beat frequency development in the wallum sedgefrog whereby there was always a strong initial increase in call frequency in reaction to the first vocal interruption by the eastern sedgefrog. These results support the hypothesis that the eastern sedgefrog and the wallum sedgefrog are in competition for the acoustic space in habitats where they occur together. This highlights a new threat to the vulnerable wallum sedgefrog species and may serve to inform future management practices. Using rhythm analyses to track changes in acoustic behavior can help inform on important population dynamics such as health, trajectory, and response to management, and therefore be of great benefit to the conservation of vocal species.

Obtaining Xenopus tropicalis Embryos by In Vitro Fertilization.

Xenopus is a powerful model system for cell and developmental biology in part because frogs produce thousands of eggs and embryos year-round. In vitro fertilization (IVF) is ideal for obtaining developmentally synchronized embryos for microinjection or when natural mating has failed to produce a fertilization. In IVF, females are induced to ovulate, and then eggs are collected by manual expression. After testes are collected from a euthanized male frog, the eggs are fertilized in vitro. The embryos are then treated with cysteine to remove the sticky protective jelly coat. Dejellied embryos are much easier to manipulate during microinjection or when sorting in a Petri dish. The jelly coat is also very difficult to penetrate with an injection needle. After microinjection, embryos are maintained in Petri dishes until desired stages are reached. Although in vitro fertilization in X. laevis and X. tropicalis is similar, critical differences in solutions, handling of testis, response of fertilized eggs directly after introduction of sperm, and developmental timing are required for successful fertilization in X. tropicalis.

Reference Intervals in Combined Veterinary Clinical Examinations of Male Black-Spotted Pond Frogs (Pelophylax nigromaculatus).

Simple SummaryThe reference intervals (RIs) of immunity, serum components, bone mineral density (BMD), and body composition in 151 males of Pelophylax nigromaculatus were established. These analyses are easily replicable and can safely and accurately diagnose the physiological condition of animals. The use of combined examination allows for the establishment of a successful conservation strategy through the identification of conservation problems in many vertebrate groups.In conservation physiology, analyzing the physiological response of an organism to understand its ability to adapt to environmental changes is a key technique in establishing a successful conservation strategy. Veterinary clinical examinations determine the physiological condition of animals accurately and safely, and this examination is synergistic when combined. The accuracy and safety of a clinical examination makes it advantageous for use in amphibians with high species diversity and numerous endangered species. However, it is necessary to establish a reference interval (RI) for precise interpretations and identification of animals with abnormalities through individual unit testing. We have established RIs for the immunity, serum components, bone mineral density (BMD), and body composition of black-spotted pond frogs (Pelophylax nigromaculatus). Black-spotted pond frogs are a common species and are widely distributed in East Asia, with suitable characteristics for environmental monitoring. Serum was extracted from 151 male frogs to establish the RI for bacterial killing ability in order to represent immunity. We also used the serum to establish an RI of ten additional serum components to determine the nutritional status, organ function status, body osmotic pressure, and homeostasis conditions. The BMD and three body composition measures for diagnosing food intake status and nutritional condition were measured using dual energy X-ray absorptiometry. The RI was recorded as the mean ± standard deviation, median, first (25%) to third (75%) quantile range, 95% confidence interval of the mean and median, and the 95% percentile (2.5%–97.5% range) of all components. The use of combined clinical veterinary examinations aids our understanding of the physiological conditions of an individual according to biotic and abiotic factors on a complex spatiotemporal scale in an ecosystem.

JNK/Elk1 signaling and PCNA protein expression in the brain of hibernating frog Pelophylax esculentus.

Mitogen activated protein kinase (MAPK) activation and neurogenesis are known to play a role in neuronal survival during hibernation. Herein, we investigate the activity of c-Jun N-terminal kinases (JNK) and Ets like-1 protein (Elk1) kinase involved in cell survival, as well as the expression of proliferating cell nuclear antigen (PCNA), a cell proliferation marker, in the brain of the frog Pelophylax esculentus. The study was conducted on female and male frogs collected during the annual cycle. Our results demonstrated that JNK activity increased during the hibernating phase in relation to the active phase. Interestingly, P-Elk1 levels were positively correlated with P-JNK levels, suggesting that the JNK/Elk1 pathway is pivotal in mediating neuroprotective adaptations that are essential to successful hibernation. On the contrary, we detected higher PCNA expression levels during the active period compared with the hibernating period. A sex dimorphism was observed in the expression levels of P-JNK/P-Elk1 that were specifically higher in males, and in the expression of PCNA reporting higher levels in female brains. Much remains to be learned regarding the regulation of hibernation, however, our findings provide new insights into the role of MAPK and proliferative pathways in hibernation, adding new knowledge concerning the mechanisms activated in the brain of ectothermic species to counteract the damage resulting from extreme temperatures.

Enigmatic Near‐Extirpation in a Boreal Toad Metapopulation in Northwestern Montana

ABSTRACTNorth America's protected lands harbor biodiversity and provide habitats where species threatened by a variety of stressors in other environments can thrive. Yet disease, climate change, and other threats are not limited by land management boundaries and can interact with conditions within protected landscapes to affect sensitive populations. We examined the population dynamics of a boreal toad (Anaxyrus boreas boreas) metapopulation at a wildlife refuge in northwestern Montana, USA, over a 16‐year period (2003–2018). We used robust design capture‐recapture models to estimate male population size, recruitment, and apparent survival over time and in relation to the amphibian chytrid fungus (Batrachochytrium dendrobatidis). We estimated female population size in years with sufficient captures. Finally, we examined trends in male and female toad body size and condition. We found no evidence of an effect of disease or time on male toad survival but detected a strong negative trend in recruitment of new males to the population. Estimates of male and female abundance decreased over time. Body size of males and females was inversely related to estimated population size, consistent with reduced recruitment to replace adults, but body condition of adult males was only weakly associated with abundance. Together, these results describe the demography of a near‐extirpation event, and point to dramatic decreases in the recruitment of new individuals to the breeding population as the cause of this decline. We surmise that processes related to the restoration of historical hydrology within the refuge adversely affected amphibian breeding habitat, and that these changes interacted with disease, life history, and other factors to restrict the recruitment of new individuals to the breeding population over time. Our results point to challenges in understanding and predicting factors that influence population change and highlight that current metrics for assessing population status can have limited predictive ability. Published 2021. This article is a U.S. Government work and is in the public domain in the USA.

Sexual selection in a tropical toad: Do female toads choose brighter males in a species with rapid colour change?

AbstractConspicuous visual signals play an important role in animal communication, both within and between species. Bright, colourful signals allow animals to discriminate between individuals and can inform behavioural decisions and social interactions. In many instances, conspicuous colouration appears to have evolved through sexual selection via female mate choice or male–male competition. Male Neotropical yellow toads, Incilius luetkenii, display bright‐yellow colouration during their brief explosive mating events and then revert to a cryptic, female‐like, brown colour following amplexus and fertilization. Recent research has revealed that the yellow colouration of males may serve as a sex identification signal and that males remain yellow in the presence of both males and females but darken without conspecific stimulus. However, there is considerable variation in the brightness of yellow colouration in courting males, ranging from dull olive‐green to vibrant lemon‐yellow. The function of this variation remains unknown. In this study, we conducted two‐choice model presentation trials to test one mechanism of sexual selection that may drive this variation: intersexual selection. Specifically, we set out to determine whether females differentiate between males based on their colouration. We presented females with two hyper‐realistic robotic model toads, with one model painted to match a bright‐yellow male and the other a dull‐yellow male. Using several metrics of choice, we found that females did not show a preference for bright or dull colouration. Our research suggests that variation in the intensity of male breeding colouration is not driven by female choice in this species. Our study is consistent with the idea that there may be limited opportunity for female choice to influence male‐trait expression in explosive breeders. Future research will address whether intrasexual selection may influence colour variation in male yellow toads.

Ex Vivo Brain Preparation to Analyze Vocal Pathways of Xenopus Frogs.

Understanding the neural basis of behavior is a challenging task for technical reasons. Most methods of recording neural activity require animals to be immobilized, but neural activity associated with most behavior cannot be recorded from an anesthetized, immobilized animal. Using amphibians, however, there has been some success in developing in vitro brain preparations that can be used for electrophysiological and anatomical studies. Here, we describe an ex vivo frog brain preparation from which fictive vocalizations (the neural activity that would have produced vocalizations had the brain been attached to the muscle) can be elicited repeatedly. When serotonin is applied to the isolated brains of male and female African clawed frogs, Xenopus laevis, laryngeal nerve activity that is a facsimile of those that underlie sex-specific vocalizations in vivo can be readily recorded. Recently, this preparation was successfully used in other species within the genus including Xenopus tropicalis and Xenopus victorianus This preparation allows a variety of techniques to be applied including extracellular and intracellular electrophysiological recordings and calcium imaging during vocal production, surgical and pharmacological manipulation of neurons to evaluate their impact on motor output, and tract tracing of the neural circuitry. Thus, the preparation is a powerful tool with which to understand the basic principles that govern the production of coherent and robust motor programs in vertebrates.

Comparison of sperm parameters and testis histological structure in lake frog (Pelophylax ridibundus) from areas with different level of anthropogenic pollution

The aim. The aim of the work was to study the indicators of spermatogenesis in the lake frog, Pelophylax ridibundus (Pallas, 1771), in the zone of the influence of emissions from the Burshtyn Thermal Power Plant (TPP).
 Materials and methods. Urinal sperm and testes samples were taken from male frogs under conditions of anthropogenic pollution (Burshtyn industrial area). Such parameters as sperm concentration in ejaculate, the number of live spermatozoa, total amount of actively mobile forms, and percentage of spermatozoa with progressive motion were determined by calculations in the hemocytometer. Testis samples were processed for routine histological method. The diameter of seminiferous tubules and histological abnormalities were studied.
 Results. Studies have revealed that near the Burshtyn TPP industrial site, the sperm concentration in the sperm sample and the total number of motile spermatozoa of the lake frogs significantly decreased. The number of dead spermatozoa increases. Amphibians from polluted sites had histological abnormalities in testes such as dilation of the seminiferous tubules with sparse sperm. At the same time, the diameter of the seminiferous tubules in the animals from polluted site significantly decreased.
 Conclusions. The impact of the emissions from the Burshtyn thermal power plant has an expressive reprotoxic effect that is manifested by depressive changes in testes tissue and gametes. The reproductive system of the lake frog is quite sensitive to environmental pollution. Sperm parameters and histological changes in testes are informative biomarkers and can be used for bioindication of technogenically transformed territories

Treefrogs adjust their acoustic signals in response to harmonics structure of intruder calls

AbstractSpectral properties of animal acoustic signals may help individuals to assess the characteristics of rivals and to adjust their competitive strategies in territorial disputes. Thus, we hypothesized that the distribution of energy across frequency bands in anuran calls determines behavioral responses in male–male competition. Using playback experiments, we investigated the relevance of the harmonic calls in the acoustic communication of the treefrog Dendropsophus minutus. We exposed territorial males to three synthetic acoustic stimuli composed of aggressive notes: 1) standard call (all harmonics and peak frequency corresponding to the second band); 2) inverted-energy call (all harmonics and peak frequency corresponding to the first band); and 3) concentrated-energy call (all energy contained in the second harmonic). Males responded aggressively to all stimuli, mainly by increasing the rate and duration of their aggressive notes. However, when exposed to stimuli with different harmonic configurations, males changed the harmonic structure of their own calls, emitting more A- and B-notes with peak power in the fundamental frequency, particularly when exposed to the concentrated-energy call. Our results suggest that male frogs may use the harmonic structure of calls to assess opponents and modulate territorial and aggressive behavior, triggering complex acoustic adjustments. This study contributes to our knowledge about the functions of acoustic traits in amphibian social interactions, and particularly of the presence of harmonics that has received less attention compared to other acoustic properties in the study of animal acoustic communication.

Efficacy of hormone stimulation on sperm production in an alpine amphibian (Anaxyrus boreas boreas) and the impact of short-term storage on sperm quality

The Southern Rocky Mountain boreal toad (Anaxyrus boreas boreas) has disappeared from much of its range in the alpine regions of Central and Western North America, and restoration efforts are compromised by limited knowledge of this species’ reproductive biology. This study aimed to establish whether assisted reproductive techniques could be used to improve breeding output in captive boreal toads by determining the most effective concentration of human chorionic gonadotropin (hCG) for induction of spermiation and viability of sperm during cold storage. Male toads (n = 21) were treated with a Low (3 IU g-1), Medium (10 IU g-1), or High (15 IU g-1) concentration of hCG and spermic urine samples were collected over 24 hrs. Treatment effectiveness was evaluated by measuring the response rate, Total Motility (TM), Forward Progressive Motility (FPM), Quality of FPM (QFPM), and concentration. For short-term cold storage, spermic urine samples (n = 13) were stored at 4 °C for 14 days and sperm TM and FPM monitored daily. All treatments induced spermiation; however, a greater number of toads produced sperm in the Medium and High treatments compared to the Low. Overall, TM, FPM, QFPM and sperm concentration were similar across all three treatments, but variation existed in the timing and duration of peak sperm production. Sperm motility was maintained for up to 14 days in cold storage, although the quality slowly decreased over time. An effective reproduction strategy for the boreal toad will provide a means to improve captive breeding efforts and increase our understanding of the reproductive physiology of alpine Bufonids.

Short-term stressors and corticosterone effects on immunity in male toads (Rhinella icterica): A neuroimmune-endocrine approach

In the last decades, it is growing the idea that stress-induced immunomodulation is bimodal: with acute stress associated with enhancing effects while chronic stress with suppressive effects. However, the immune-endocrine interactions and its implications are often overlooked in ectotherms. We investigated the impact of corticosterone (CORT) treatment and short-term stressors on CORT plasma levels and the immunity of male toads (Rhinella icterica), using three distinct protocols: restraint, immune challenge (with lipopolysaccharide, LPS), and CORT transdermal application (TA). Our results showed increased CORT and neutrophil: lymphocyte ratio (NLR) regardless of the stress input (restraint, LPS challenge) or CORT TA. In the meantime, the bacterial killing ability (BKA) was not affected by any treatment, suggesting this immune parameter might be a more constitutive and robust response. Interestingly, the cellular immune response showed distinct patterns. Increased phagocytosis of blood leukocytes and phytohemagglutinin edema followed LPS and CORT TA (15 ​μg), respectively. In contrast, the phagocytosis of peritoneal leukocytes decreased after CORT TA (1 and 10 ​μg), indicating that short-term increases in CORT levels might impair local immune function. Such differences in cellular immunity might also be associated with CORT doses or the interaction between CORT and other immune mediators, such as melatonin, testosterone, and cytokines. Overall, our results highlight the immune-enhancing effects of the acute stress response and CORT TA, and the complexity of the immune-endocrine interaction in anurans. It also highlights the relevance of investigating distinct contexts for CORT increase arising from different situations, as well as diverse immune components for a better understanding of the stress-induced immunomodulation.

The development of method of hormonal stimulation of N.L.Gerbilsky and appearance of contemporary preparations of long-term storage

The The main discovery of the 20th century in the field of fish breeding is hormonal stimulation of final stages of maturation of fish at hatcheries. The «father» of method of pituitary injections was Leningrad ichthyologist N.L. Gerbilsky. The main achievements of N.L. Gerbilsky and his school are methods of mass extraction of fish pituitaries, their dehydration by acetone for later storage and use, testing of pituitary gonadotropic activity on different objects. The induction of ovulation in female loaches Misgurnus fossilis or spermiation reaction of Galli-Mainini in male frogs Rana ridibunda or R. temporaria were used for pituitary testing. The effective doses of dried pituitaries for sturgeons were determined in milligrams and loach units or frog units. The method of pituitary injections was used for functioning of sturgeon breeding facilities at Volga, Don, Kuban and Kura and other rivers. For guaranteed answer fish breeders used hyperdoses of dry pituitary powder because its gonadotropic activity varied in dependence on stage of maturity of fish-“donors”. The followers of N.L. Gerbilsky led by Prof. I.A. Barannikova developed the technique of production of special glycerol pituitary extract (GPE) with determined gonadotropic activity. The main role in formulation of the recipe of GPE belongs to A.A. Boyev. The use of GPE has removed the problem of hyperdosing leading to worsening quality of mature germinal cells of sturgeons. Later, A.A. Boyev on the basis of this recipe with replacement of glycerol to propylene glycol created stabilizing solution of analogue of mammal LH-RH (“surphagon”) of long-term storage with concentration of active substance from 20 to 500 micrograms/ml.

Calling activity determines male mating success in a territorial frog with parental care

AbstractA contentious aspect in dendrobatoid frog behaviour is the recognition of the predominant factor—if any—driving male mating success. This is probably related to an extensive correlation among different male quality predictors. Here, we investigated the diurnal and territorial nurse frog Allobates subfolionidificans by means of a field study during one entire breeding season in Brazilian Amazonia. We tested the roles of two intrinsic male properties (body size and calling activity) and five territory‐related variables (territory size, vegetation cover, nesting site, perch height and food availability) on male reproductive output. A multiple regression analysis revealed calling activity as the only variable predicting male mating success in A. subfolionidificans. Vocalisation probably represents the most energetically expensive activity that a male frog undertakes during his lifetime, thus calling activity may be used by A. subfolionidificans females as a cue to mate choice that indicates competitive potential and male quality. Alternatively, non‐choosy females also should be more likely to encounter and mate with males that advertise more often throughout the breeding season.