Breeding Glands Research Articles

Sexy fingers: Pheromones in the glands of male dendrobatid frogs.

Many animals exchange chemicals during courtship and mating. In some amphibians, sexual chemical communication is mediated by pheromones produced in male breeding glands that are transferred to the female's nostrils during mating. This has been mostly studied in salamanders, despite frogs having similar glands and courtship behaviours suggestive of chemical communication. In Neotropical poison frogs (Dendrobatidae and Aromobatidae), males of many species develop breeding glands in their fingers, causing certain fingers to visibly swell. Many also engage in cephalic amplexus, whereby the male's swollen fingers are placed in close contact with the female's nares during courtship. Here, we investigate the possible roles of swollen fingers in pheromone production using whole-transcriptome sequencing (RNAseq). We examined differential gene expression in the swollen versus non-swollen fingers and toes of two dendrobatid species, Leucostethus brachistriatus and Epipedobates anthonyi, both of which have specialised mucous glands in finger IV, the latter of which has cephalic amplexus. The overwhelming pattern of gene expression in both species was strong upregulation of sodefrin precursor-like factors (SPFs) in swollen fingers, a well-known pheromone system in salamanders. The differentially expressed SPF transcripts in each species were very high (>40), suggesting a high abundance of putative protein pheromones in both species. Overall, the high expression of SPFs in the swollen fingers in both species, combined with cephalic amplexus, supports the hypothesis that these traits, widespread across members of the subfamilies Colostethinae and Hyloxalinae (ca. 141 species), are involved in chemical signalling during courtship.

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.

Transcriptome analysis of anuran breeding glands reveals a surprisingly high expression and diversity of NNMT-like genes

Abstract In many amphibians, males have sexually dimorphic breeding glands, which can produce proteinaceous or volatile pheromones, used for intraspecific communication. In this study we analyse two types of glands in the Mexican treefrog Ptychohyla macrotympanum (Hylidae)—large ventrolateral glands and small nuptial pads on their fingers—using histology, whole-transcriptome sequencing, and phylogenetic analyses. We found strong differences in glandular tissue composition and gene expression patterns between the two breeding gland types. In both glands we only found low expression of protein pheromone candidates. Instead, in the ventrolateral glands, gene expression was strikingly dominated by nicotinamide N-methyltransferase (NNMT)-like genes. The diversity of these genes was remarkably high, with at least 68 distinct NNMT-like genes. Our comparative phylogenetic analysis of the diversity of NNMT-like genes across vertebrates indicates that the extreme diversity of this gene is largely a frog-specific phenomenon and can be traced to large numbers of relatively recent gene duplications occurring independently in many lineages. The strong dominance and astonishing diversity of NNMT-like genes found in anurans in general, and in their sexually dimorphic breeding glands specifically, suggests an important function of NNMT-like proteins for anuran reproduction, possibly being related to volatile pheromone production.

Pheromones for amphibian conservation – considering new approaches in conservation breeding programmes

Pheromones for amphibian conservation – considering new approaches in conservation breeding programmes

Gene expression of secretory proteins in the nuptial pads of three Lithobates species (Anura: Ranidae)

Abstract Many amphibian species possess male-specific secretory breeding glands. In anurans, such specialized glands are often present as nuptial pads at the first digit of the hand, which are pressed against the female during amplexus. However, the role of nuptial pad secretions remains largely unknown. Here, we investigate the inner morphology as well as the RNA expression patterns of the nuptial pads of several Central American Lithobates species (Ranidae). As shown for the breeding glands of other amphibian species, the Lithobates nuptial pads are composed of large specialized mucus glands, excreting proteinaceous content to the surface. Whole-transcriptome sequencing revealed that for one of the species the most highly expressed transcripts encoding secretory proteins in the nuptial pads are sodefrin precursor-like factor (SPF) proteins, known to have a pheromone function in multiple amphibian species. The other two species, however, lack high expression of SPF transcripts but express other secretory proteins, whose roles in amphibian breeding glands remain unknown. Several of these proteins are highly expressed in all samples, suggesting a direct role during courtship or for the maintenance/development of the glands.

Love bites: male frogs (Plectrohyla, Hylidae) use teeth scratching to deliver sodefrin precursor-like factors to females during amplexus

BackgroundEfficient transfer of chemical signals is important for successful mating in many animal species. Multiple evolutionary lineages of animals evolved direct sex pheromone transmission during traumatic mating—the wounding of the partner with specialized devices—which helps to avoid signal loss to the environment. Although such direct transmission modes of so-called allohormone pheromones are well-documented in invertebrates, they are considered rare in vertebrates. Males of several species of the frog genus Plectrohyla (Hylidae, Anura) have elongated teeth and develop swollen lips during the breeding season. Here we investigated the possibility that these structures are used to scratch the females’ skin and apply allohormone pheromones during traumatic mating in several Plectrohyla species.ResultsOur behavioural observations revealed that males press their upper jaw onto the females’ dorsum during amplexus, leaving small skin scratches with their teeth. Histological examinations of the males’ lips identified specialized mucus glands, resembling known amphibian pheromone glands. Whole-transcriptome sequencing of these breeding glands showed high expression of sodefrin precursor-like factor (SPF) proteins, which are known to have a pheromone function in multiple amphibian species.ConclusionsOur study suggests SPF delivery via traumatic mating in several anuran species: the males have specialized breeding glands in the lips for production and secretion and use their elongated teeth as wounding devices for application. We hypothesize that these SPF proteins end up in the females’ circulatory system, where understanding their exact function will require further molecular, physiological and behavioural testing.

Sexually dimorphic skin glands in the invasive species Lithobates catesbeianus (Anura: Ranidae)

Abstract Males of most amphibian species possess specialized cutaneous glands, known as sexually dimorphic skin glands (SDSGs). SDSGs are usually clustered in specific body regions and are externally visible, but in some cases, external differences between males and females can be slight or absent, and the occurrence of SDSGs can only be disclosed by histological studies. Chemical signals produced by SDSGs markedly affect amphibian behaviour and reproduction, and therefore their occurrence, features, and location in the body could provide information on potential mechanisms of intraspecific communication in a particular species. In the present study, we perform light microscope (both histological and histochemical), and scanning electron microscope studies of skin samples from male and female adult specimens of the invasive bullfrog Lithobates catesbeianus, covering several body regions that could hold SDSGs. Most skin areas analysed showed only ordinary granular and mucous glands despite remarkable sexual dimorphism that could be externally observed. By contrast, the male nuptial pads contained exclusively SDSGs that were hypertrophied specialized mucous glands (SMGs), closely resembling breeding glands described in other anurans. Our histochemical study revealed that these SMGs contain heterogeneous populations of secretory cells, possibly involved in pheromone production. We discuss these characteristics of the SDSGs found in L. catesbeianus, as well as the surface specialization of the nuptial pads (achieved by scanning electron microscopy) in the light of their potential role in the chemical communication in this invasive species.

High pheromone diversity in the male cheek gland of the red-spotted newt Notophthalmus viridescens (Salamandridae).

BackgroundMale salamanders (Urodela) often make use of pheromones that are produced in sexually dimorphic glands to persuade the female into courtship and mating. The mental gland of lungless salamanders (Plethodontidae) and dorsal cloacal glands (or abdominal glands) of newts (Salamandridae) have been particularly well studied in that respect. In both families, sodefrin precursor-like factor (SPF) proteins have been identified as major components of the courtship pheromone system. However, similar to plethodontids, some newts also make use of subtle head glands during courtship, but few pheromones have been characterized from such structures. Males of red-spotted newts (Notophthalmus viridescens, Salamandridae) have both cloacal and cheek (genial) glands, and are known to apply secretions to the female’s nose by both tail-fanning and cheek-rubbing. Here we combined transcriptomic and phylogenetic analyses to investigate the presence, diversity and evolution of SPF proteins in the cloacal and cheek glands of this species.ResultsOur analyses indicate that the cheek glands of male N. viridescens produce a similar amount and diversity of SPF isoforms as the cloacal glands in this species. Expression in other tissues was much lower, suggesting that both male-specific courtship glands secrete SPF pheromones during courtship. Our phylogenetic analyses show that N. viridescens expresses a combination of isoforms that stem from four highly diverged evolutionary lineages of SPF variants, that together form a basis for the broad diversity of SPF precursors in the breeding glands.ConclusionsThe similar SPF expression of cheek and cloacal glands suggests that this protein family is used for pheromone signalling through cheek rubbing in the red-spotted newt. Since several male salamandrids in other genera have comparable head glands, SPF application via other glands than the cloacal glands may be more widespread than currently appreciated in salamandrids.Electronic supplementary materialThe online version of this article (doi:10.1186/s12862-015-0333-1) contains supplementary material, which is available to authorized users.

Seasonal lectin binding variations of thumb pad in the frog (Pelophylax ridibundus)

The thumb pad is one of the most common secondary sexual characteristics in frogs. Although it is known that amphibian skin has affinity for several lectins, there is no report regarding lectin-binding affinity of the thumb pad or its structural components. This study investigated localization and seasonal variation of specific carbohydrate moieties of glycoconjugates in both the epidermal and dermal components of the frog thumb pad at the light microscopic level using lectin histochemistry. The study consisted of four seasonal groups of the frog species, Pelophylax ridibundus (Synonym of Rana ridibunda): active, prehibernating, hibernating and posthibernating. Four horseradish peroxidase conjugated lectins were employed. It was found that dolichos biflorus agglutinin (DBA), wheat germ agglutinin (WGA), and ulex europaeus (UEAI) gave positive reactions in both epidermal layers and breeding glands. These three lectins bound specific secretory cells in the breeding glands, and the distribution of the cells and epithelial lectin reactions exhibited seasonal changes. In addition, UEA-I and peanut agglutinin (PNA) showed an affinity in granular glands and the granular zone of mixed glands. Generally, epidermal lectin binding showed dense affinity during the posthibernation period. DBA, UEA-I, and WGA-specific cells in the mucous gland decreased gradually until the posthibernation period. These findings suggest that differences of lectin binding in the thumb pad may be related to functional activities and, thus, seasonal adaptations. Moreover, the presence of specific lectin-binding cells in the breeding glands indicated that they consisted of heterogeneous secretory cell composition or that the cells were at different secretory stages.

Structural diversity of nuptial pads in Phyllomedusinae (Amphibia: Anura: Hylidae)

We studied the morphological variation of the nuptial pads using light microscopy and scanning electron microscopy (SEM) in 26 species of phyllomedusines (Anura: Hylidae), representing the five currently recognized genera. All phyllomedusines have single nuptial pads with dark colored epidermal projections (EPs). Spine-shaped EPs occur in Cruziohyla calcarifer, Phrynomedusa appendiculata and in one species of Phasmahyla. The other species have roundish EPs. The density of the EPs on the pad is variable. Species in the Phyllomedusa hypochondrialis Group have EPs with a density that varies between 764 ± 58/mm(2) and 923 ± 160/mm(2). In all other studied species (including the Phyllomedusa burmeisteri and Phyllomedusa perinesos groups, Phyllomedusa camba, Phyllomedusa boliviana, Phyllomedusa sauvagii, Phyllomedusa bicolor, and Phyllomedusa tomopterna) the density of EPs varies between 108 ± 20/mm(2) and 552 ± 97/mm(2). Pores were observed with SEM in C. calcarifer, Agalychnis lemur, Agalychnis moreletii, but its presence is confirmed through histological sections on several other species. Its visibility using SEM seems to be related with the level of separation between adjacent EPs. The pores in the four studied species of Agalychnis are shown with SEM and histological sections to have a characteristic epidermal rim, that is absent in the otherphyllomedusines. Unlike most previous reports on breeding glands, those of phyllomedusines are alcian blue positive, indicating the presence of acidic mucosubstances on its secretions.

Ultrastructure and Histochemistry of the Adhesive Breeding Glands in Male Gastrophryne carolinensis (Amphibia: Anura: Microhylidae)

The histology, histochemistry, and ultrastructure of the adhesive breeding glands of male Gastrophryne carolinensis are described. Adhesive glands are mutlicellular exocrine glands in the dermis of the sternum and forearm that cause the male to adhere to the female during amplexus. The epithelial cells have distinct plasma membranes, and the product consists of electron-dense secretory granules that fill the cytoplasm and are released intact by an apocrine process. We support one previous study and contradict another report by finding that adhesive glands react positively for neutral carbohydrates and negatively for glycosaminoglycans and proteins. The ultrastructural results, the first on these organs, confirm that adhesive glands are derived from mucous glands and not serous glands.

Annual Morphological Cycles of Testis and Thumb Pad of the Male Frog (Rana ridibunda)

In this study, Rana ridibunda was used as samples because of their wide use in Turkey. Male frogs were collected in the East Marmara region each month throughout 1 year. Frogs from every monthly collection were used to analyze structural components of the thumb pads and testes. Spermatogenetic activity of Rana ridibunda living in the East Marmara region was determined to be "potentially continuous" type. Generally, the increase in the number and the size of nuclei of Leydig cells was inversely proportional to the fluctuation of spermatogenetic activity. The lumen of the seminiferous tubules in testes contained, in addition to the spermatogenic cells, a Periodic-acid Schiff-positive granular material. The amount of this material varied throughout the year, and that finding suggested a function related to spermiation. The components of thumb pads exhibited structural changes with respect to the activities of Leydig cells. During the periods where the Leydig cells were active, mucus glands (also called breeding glands) of thumb pads were also developed. On the other hand, we observed mixed glands with unknown function, which as first reported by us, and were poison glands in the thumb pads. The results suggest structural changes in the thumb pads are linked to changes in the testes.

The effects of anti-androgenic and estrogenic disrupting contaminants on breeding gland (nuptial pad) morphology, plasma testosterone levels, and plasma vitellogenin levels in male Xenopus laevis (African clawed frog).

Nuptial pads are areas, digits, and forearm characterized by epidermal keratin hooks and dermal breeding glands opening on the epidermis surface in male frogs. The presence of the epidermal hooks and secretory activity of the breeding glands are androgen-dependent and considered functioning during mating. Chemical pollutants released into the aquatic environment by humans are suspected of disrupting the normal hormonal pathways and functioning. Among the best known of these endocrine-disrupting contaminants (EDCs) are compounds that mimic the steroid 17beta-estradiol. However, recently specific anti-androgenic activity by certain EDCs, including DDE and several fungicides, have been shown in mammalian studies. The African clawed frog, Xenopus laevis, has been proposed as an ideal species to be used as a model system for screening of endocrine disruptors. In this study we employed a newly developed ELISA for the measurement of plasma vitellogenin (VTG), normally produced in the liver of females under the influence of estrogens but also in males when exposed to exogenous estrogens. Furthermore, we investigated the potential of breeding (nuptial) gland activity to be employed as a biomarker system for screening for anti-androgenic activity by certain EDCs. Our results indicate that the pharmaceutic anti-androgen flutamide did significantly (p < 0.05) effect the androgen-dependent breeding (nuptial) glands and plasma testosterone concentrations in male X. laevis. Our results further confirm that the dicarboximide fungicide vinclozolin mimics the anti-androgenic action of flutamide. Vinclozolin, however, did not significantly effect the plasma testosterone concentration. Results also confirm the ability of 17beta-estradiol to stimulate production and release of VTG in the liver of male X. laevis, although we could not confirm the in vivo induction of VTG by estrogenic mimics octylphenol and nonylphenol. Although more dose-response research is needed, this preliminary study confirms the potential of VTG production and breeding gland activity as biomarkers to screen chemicals and environmental samples for estrogenic and anti-androgenic activity using X. laevis males.

Evidence for a Mate-Attracting Chemosignal in the Dwarf African Clawed Frog Hymenochirus

Many male frogs and toads possess sexually dimorphic skin glands (breeding glands). However, in most anuran species, the functional significance of the glands is unknown. Here we show that the breeding glands of male dwarf African clawed frogs (Hymenochirus sp.) release a mate-attractant chemosignal. The mate-attractant activity was assessed using a two-choice aquatic Y-maze. Female Hymenochirus were allowed to choose between different treatment waters (e.g., plain water and water housing males) in the upstream arms of the maze, and the females' movements were monitored by computer-linked motion sensors. Females showed a positive chemotaxis to water housing males and to water containing homogenized breeding glands. Females showed no reaction to water housing conspecific females or to water housing breeding gland-ablated males. Additional choice tests demonstrated that females were more attracted to water housing males than to water housing females and to water containing homogenized breeding glands than to water housing breeding gland-ablated males. Males in the maze showed no response to water housing either females or other males, indicating that the attractant is specific for females and is therefore neither a species aggregation signal nor a food-related attractant. These results represent the first experimental demonstration of a mate-attractant function for anuran breeding glands. Because many anuran species possess breeding glands, these results suggest that pheromonal communication could be more widespread among frogs and toads than previously believed.

Testicular and androgen dependence of skin gland morphology in the anurans, Xenopus laevis and Rana pipiens.

In anuran amphibians, there is increasing evidence that exocrine glands dispersed throughout the general integument are secondary sex characters (SSC). Following the recent discovery of sexually dimorphic "breeding glands" in the dorsum of male Rana pipiens, we studied the effects of castration and testosterone treatment on the dorsal skin glands of male Xenopus laevis and R. pipiens to determine whether the dorsal breeding glands, or any other dorsal skin glands, are androgen dependent. The dorsal skin glands of X. laevis were unaffected by androgen status. By contrast, in R. pipiens, breeding, mucous, and seromucous glands responded to testosterone stimulation. Mucous glands were significantly (P < 0.05) larger in testosterone-treated frogs than in castrates. There was a large, but statistically insignificant, increase in the size of the dorsal breeding glands. Testosterone treatment also increased the epithelial cell height of breeding and seromucous glands (P < 0.05). In the skins of castrated and testosterone-treated frogs, there was a reciprocal relationship between the abundance of seromucous and breeding glands: in castrates, seromucous glands were abundant and breeding glands virtually absent, whereas in testosterone-treated frogs, breeding glands were abundant and seromucous glands less common. The total number of the two gland types was similar in both treatment groups. Glands that appeared to be intermediate in form between seromucous and breeding glands were observed in some frogs. These data suggest that seromucous glands may be the regressed form of breeding glands in the dorsal skin of R. pipiens and that the dorsal skin of R. pipiens is a SSC. © 1993 Wiley-Liss, Inc.

The Influence of Hormones on the Development of Breeding Glands in Microhyla

The Influence of Hormones on the Development of Breeding Glands in Microhyla