A unique mating strategy without physical contact during fertilization in Bombay Night Frogs (Nyctibatrachus humayuni) with the description of a new form of amplexus and female call.

Anurans show the highest diversity in reproductive modes of all vertebrate taxa, with a variety of associated breeding behaviours. One striking feature of anuran reproduction is amplexus. During this process, in which the male clasps the female, both individuals' cloacae are juxtaposed to ensure successful external fertilization. Several types of amplexus have evolved with the diversification of anurans, and secondary loss of amplexus has been reported in a few distantly related taxa. Within Nyctibatrachus, a genus endemic to the Western Ghats of India, normal axillary amplexus, a complete loss of amplexus, and intermediate forms of amplexus have all been suggested to occur, but many species remain unstudied. Here, we describe the reproductive behaviour of N. humayuni, including a new type of amplexus. The dorsal straddle, here defined as a loose form of contact in which the male sits on the dorsum of the female prior to oviposition but without clasping her, is previously unreported for anurans. When compared to known amplexus types, it most closely resembles the form of amplexus observed in Mantellinae. Furthermore, we prove that, opposed to the situation in most anurans, male semen release happens before egg deposition. We hypothesize that the male ejaculates on the female's dorsum and that sperm subsequently runs from her back and hind legs before fertilizing the eggs. A second feature characterizing anuran breeding is the advertisement call, mostly produced solely by males. Despite recent descriptions of several new Nyctibatrachus species, few studies have explored their vocal repertoire. We describe both the male advertisement call and a female call for N. humayuni. The presence of a female call has not been reported within Nyctibatrachidae, and has been reported in less than 0.5% of anuran species. Altogether, our results highlight a striking diversity and several unique aspects of Nyctibatrachus breeding behaviour.

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

BACKGROUND: Male 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. RESULTS: Our 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. CONCLUSIONS: The 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.

Side-by-side secretion of Late Palaeozoic diverged courtship pheromones in an aquatic salamander.

Males of the advanced salamanders (Salamandroidea) attain internal fertilization without a copulatory organ by depositing a spermatophore on the substrate in the environment, which females subsequently take up with their cloaca. The aquatically reproducing modern Eurasian newts (Salamandridae) have taken this to extremes, because most species do not display close physical contact during courtship, but instead largely rely on females following the male track at spermatophore deposition. Although pheromones have been widely assumed to represent an important aspect of male courtship, molecules able to induce the female following behaviour that is the prelude for successful insemination have not yet been identified. Here, we show that uncleaved sodefrin precursor-like factor (SPF) protein pheromones are sufficient to elicit such behaviour in female palmate newts (Lissotriton helveticus). Combined transcriptomic and proteomic evidence shows that males simultaneously tail-fan multiple ca 20 kDa glycosylated SPF proteins during courtship. Notably, molecular dating estimates show that the diversification of these proteins already started in the late Palaeozoic, about 300 million years ago. Our study thus not only extends the use of uncleaved SPF proteins outside terrestrially reproducing plethodontid salamanders, but also reveals one of the oldest vertebrate pheromone systems.

Origin and diversification of a salamander sex pheromone system.

Sex pheromones form an important facet of reproductive strategies in many organisms throughout the Animal Kingdom. One of the oldest known sex pheromones in vertebrates are proteins of the Sodefrin Precursor-like Factor (SPF) system, which already had a courtship function in early salamanders. The subsequent evolution of salamanders is characterized by a diversification in courtship and reproduction, but little is known on how the SPF pheromone system diversified in relation to changing courtship strategies. Here, we combined transcriptomic, genomic, and phylogenetic analyses to investigate the evolution of the SPF pheromone system in nine salamandrid species with distinct courtship displays. First, we show that SPF originated from vertebrate three-finger proteins and diversified through multiple gene duplications in salamanders, while remaining a single copy in frogs. Next, we demonstrate that tail-fanning newts have retained a high phylogenetic diversity of SPFs, whereas loss of tail-fanning has been associated with a reduced importance or loss of SPF expression in the cloacal region. Finally, we show that the attractant decapeptide sodefrin is cleaved from larger SPF precursors that originated by a 62 bp insertion and consequent frameshift in an ancestral Cynops lineage. This led to the birth of a new decapeptide that rapidly evolved a pheromone function independently from uncleaved proteins.

Frog nuptial pads secrete mating season-specific proteins related to salamander pheromones.

Males of many frog species develop spiny nuptial pads with underlying glands on their thumbs during the mating period. We used 3D visualization on the European common frog Rana temporaria to show that the morphology of these glands allows the channelling of secreted molecules to the pad's surface during amplexus. Combined transcriptome and proteome analyses show that proteins of the Ly-6/uPAR family, here termed amplexins, are highly expressed in the nuptial glands during the mating season, but are totally absent outside that period. The function of amplexins remains unknown, but it is interesting to note that they share structural similarities with plethodontid modulating factors, proteins that influence courtship duration in salamanders.

Love is blind: indiscriminate female mating responses to male courtship pheromones in newts (Salamandridae).

Internal fertilization without copulation or prolonged physical contact is a rare reproductive mode among vertebrates. In many newts (Salamandridae), the male deposits a spermatophore on the substrate in the water, which the female subsequently takes up with her cloaca. Because such an insemination requires intense coordination of both sexes, male newts have evolved a courtship display, essentially consisting of sending pheromones under water by tail-fanning towards their potential partner. Behavioral experiments until now mostly focused on an attractant function, i.e. showing that olfactory cues are able to bring both sexes together. However, since males start their display only after an initial contact phase, courtship pheromones are expected to have an alternative function. Here we developed a series of intraspecific and interspecific two-female experiments with alpine newt (Ichthyosaura alpestris) and palmate newt (Lissotriton helveticus) females, comparing behavior in male courtship water and control water. We show that male olfactory cues emitted during tail-fanning are pheromones that can induce all typical features of natural female mating behavior. Interestingly, females exposed to male pheromones of their own species show indiscriminate mating responses to conspecific and heterospecific females, indicating that visual cues are subordinate to olfactory cues during courtship.

Mountain-associated clade endemism in an ancient frog family (Nyctibatrachidae) on the Indian subcontinent.

Night frogs (Nyctibatrachidae) form a family endemic to the Western Ghats, a hill chain along the west coast of southern India. Extant members of this family are descendants of a lineage that originated on the subcontinent during its longtime isolation in the Late Cretaceous. Because the evolutionary history of Nyctibatrachidae has always been tightly connected to the subcontinent, these tropically-adapted frogs are an ideal group for studying how patterns of endemism originated and evolved during the Cenozoic in the Western Ghats. We used a combined set of mitochondrial and nuclear DNA fragments to investigate the phylogenetic relationships of 120 ingroup specimens of all known species of Nyctibatrachidae. Our analyses indicate that, although this family had an early origin on the Indian subcontinent, the early diversification of extant nyctibatrachids happened only in the Eocene. Biogeographic analyses show that dispersal across the Palghat gap and Shencottah gap was limited, which led to clade endemism within mountain ranges of the Western Ghats. It is likely that multiple biota have been affected simultaneously by these prominent geographical barriers. Our study therefore further highlights the importance of considering the Western Ghats-Sri Lanka biodiversity hotspot as an assemblage of distinct mountain regions, each containing endemism and deserving attention in future conservation planning.

In vivo soft tissue damage assessment for applications in surgery.

In robotic and conventional minimally invasive surgery the risk of complications caused by collateral tissue damage remains high. This paper studies the concept of imposing damage thresholds on surgical instruments to avoid tissue overload. More specifically, the correlation between mechanical loading and damage in case of vascular clamping is investigated. With a computer controlled device, a high and a low clamping load were applied in vivo on the abdominal aorta of 43 rats. Samples of both loading levels were compared with zero load control samples and with samples clamped by a mosquito clamp w.r.t. functionality and histological integrity. Analysis of the samples shows that high clamping forces result in endothelial and smooth muscle cell destruction. Clamping with a mosquito clamp will cause even more damage to the elastic lamellae. Samples loaded at the lower load showed significantly less smooth muscle cell damage and a lower degree of endothelial damage. This paper is the first to statistically quantify the correlation between the degree of mechanical loading and the degree of tissue damage, thus setting the first steps towards tissue overload prevention during surgery. Future experiments will also include the effects of loading duration, recovery and patient-specificity.

Correlation between compression, tensile and tearing tests on healthy and calcified aortic tissues.

An anastomosis performed in calcified tissues tears up faster than in healthy tissues. This study develops and validates an in vitro non-destructive method to distinguish healthy from calcified aortic tissues. An uniaxial unconfined compression test is able to distinguish healthy from calcified aortas (p<0.01). The compressive E-modulus at a strain level of 10% is 227+/-34kPa for artificially calcified and 147+/-15kPa for healthy porcine aortic tissues. Calcified aortic tissues have a lower tensile strength than healthy porcine aortic tissues (p<0.05). The ultimate tensile strength is 1.34+/-0.18MPa and 1.55+/-0.31MPa for artificially calcified and healthy porcine aortic tissues respectively. Calcified aortic tissues have a lower resistance to tearing than healthy aortic tissues (p<0.05). The resistance to tearing is 1.78+/-0.33N/mm and 2.16+/-0.64N/mm for artificially calcified and healthy porcine aortic tissues respectively.