Search

Additional file 7. Overview of obp genes with previously identified secretion sites of their corresponding proteins.

Additional file 1. Information on individual dogs and total protein concentrations of ASGS samples.

Males of advanced salamanders (Salamandroidea) are unique among vertebrates in having internal fertilization without copulation. Instead, sperm is transferred via an external spermatophore, which the female takes up with her cloaca. Since a successful transfer of sperm heavily relies on a female following the male track at spermatophore deposition, males have evolved various courtship displays to persuade a female in picking up the sperm. In several aquatically reproducing newts, males do not longer display physical contact during courtship, but instead rely on emitting chemosignals to the female. Previously, we isolated and experimentally tested protein pheromones of the Sodefrin Precursor-like Factor (SPF) family and demonstrated that they can elicit female courtship responses in a European newt. Here we combined proteomic analyses with transcriptome analyses (RNASeq) of the pheromone-producing gland to show that several European newt species additionally secrete multiple 15 kDa proteins in the water duringcourtship. These cysteine-rich proteins do not show any apparent relationship to other known proteins in vertebrates and are, similar to SPF, among the most highly expressed proteins in the pheromone-producing glands. This high expression, together with abundant secretion during tail-fanning in water, suggests that the 15 kDa proteins serve a courtship pheromone function in newts.

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, since 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 been identified yet. 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 demonstrates 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.

Internal fertilization without copulation or another form of prolonged physical contact is a rare reproductive mode among vertebrates. In newts (Salamandridae), the male deposits a spermatophore on the substrate in the water, which the female subsequently takes up with her cloaca. Such an insemination without amplexus requires intense coordination of both sexes. Male newts have evolved a courtship display, essentially consisting of tail-waving towards their partner under water. The female reacts by following the male, and the latter leads the female over the spermatophore, which adheres to her cloaca. Although this courtship has been first described nearly two centuries ago, and the involvement of both visual and olfactory stimuli has been proposed, the exact effect of male tail-waving on females remains obscure. Pheromone experiments until now focused on attraction and therefore don't provide a function for courtship at close distance. Here we developed a series of experiments comparing behavior in courtshipwater and control water in newts. We show that species-specific male pheromones emitted during tail-waving can induce all typical features of natural female mating behavior and therefore are the key to successful reproduction without physical contact.

After hibernation on land, European newts (Salamandridae, Amphibia) return to the water for courtship and breeding. Males develop several sexual characteristics such as a bright coloration and an extended crest on back and tail. The tail is used for fanning (underwater waving) and is predicted to direct pheromones from the male cloaca to the nose of the female in a complex courtship behaviour. Although this behaviour has been known for decades, the molecules expressed during waving and their exact function have never been demonstrated. We studied the courtship of palmate newts (Lissotriton helveticus) in an integrative approach, combining ethological tests, transcriptomics and proteomics. First, we tested the ability of complete courtship water (water in which males just fanned their tail) to attract females and obtained a positive behavioural assay. To further characterise the molecules responsible for this attraction, we then isolated peptides and proteins from the courtship water and used precursor cDNA information to fully characterise them. Our analyses identified multiple peptides and proteins up to 200 amino acids, showing that newt courtship water contains more than a single pheromone, and likely holds multiple kinds of chemical information.

Males of aquatic salamandrid newts often use tail-fanning behavior to direct cloacal pheromones towards receptive females, but few of these molecules have been characterized. The Asian Cynops pyrroghaster is known to emit a pheromone called sodefrin, a decapeptide that is cleaved from the larger Sodefrin Precursor Factor (SPF). Behavioral tests have demonstrated that sodefrin has the ability to attract and excite gravid females. To understand the evolution of SPF and pinpoint the origin of sodefrin, we sequenced cloacal mRNA (transcribed DNA) from males and females in five salamandrid genera with aquatic courtship. We found a substantial difference in number of transcripts between the sexes, and recovered up to seventeen different SPF transcripts per species. Integration of genome and transcriptome analyses reveal that this SPF transcript diversity is formed by a combination of gene/exon duplications and alternative splicing. Phylogenetic analyses further indicate that sodefrin evolved from SPF by a frameshift mutation within the lineage of Asian newts, i.e. long after the origin of the tail fanning behavior. We hypothesize that other salamandrid newts are using the same behavior to deliver different pheromones that are likely derived from similar SPF precursors.

During courtship, male newts of the family Salamandridae wave their tail to channel cloacal pheromones towards receptive females. In the Asian genus Cynops, this pheromone is a decapeptide called sodefrin, which is cleaved from a larger precursor protein called SPF (Sodefrin Precursor Factor). Behavioral tests have demonstrated that sodefrin has the ability to attract and excite gravid females. To understand the evolution of SPF in Salamandridae, we sequenced cloacal SPF mRNA from sexually active males and females of six species in five genera with an aquatic courtship. We found substantial differences between sexes, and recovered up to seventeen unique SPF transcripts per species encoding fundamentally different protein structures. Phylogenetic analyses indicate that this diversity arose by duplication events that happened both before and during the radiation of these aquatic salamander genera. Structural variation not only originated by DNA substitutions, but also by a frameshift mutation and by the gain and loss of alternative peptide cleavage sites. Our findings indicate that SPF represents a fast-evolving protein complex that may have contributed more to the evolution of salamandrid pheromone communication than currently thought.

Sex pheromones are chemical signals that are used in exchanging sexual messages between individuals of the same species. These molecules can serve multiple functions, including the attraction of mates, the discrimination between mate quality levels and the induction of ovulation in females. Here we used transcriptome and peptidome techniques to identify candidate sex pheromones in two aquatic courting frogs. In combination with ethological tests, we demonstrate the presence of several sex pheromone homologues in anurans. Phylogenetic analyses suggest a high degree of intraspecific differentiation, and additionally indicate multiple gene duplications during anuran evolution. These ancient duplication events may have played an important role in the evolution of anuran pheromone communication.

In search of pheromones in the amphibian skin Raepsaet A.1, Roelants K.1, Janssenswillen S.1, Clijnen E.2, Bossuyt F.1 1Departement Biology, VUB, Brussel, Belgium. 2Departement 'Functional genomics & proteomics, KUL, Leuven, Belgium Keywords: frogs, pheromone, antimicrobial peptide Most studies on amphibian skin peptides have an explicit pharmacological focus, and the origin, diversity, and functional diversification of these molecules therefore remain poorly understood. Antimicrobial peptide research in these animals has been restricted to relatively few closely related genera in a limited number of families. Furthermore, although behavioural tests suggest chemical communication during courtship in many amphibian species, only a single pheromone peptide has been characterized in anurans (frogs and toads), and only three in caudates (salamanders and newts). Here we integrated transcriptome sequencing, peptidome analyses, functional assays, and phylogenetic analyses to identify and characterize novel antimicrobial and pheromone peptides in sexually dimorphic glands of two frog species. Comparative peptidomics resulted in the identification of several male-specific molecules, which are thus identified as candidate pheromones. Preliminary behavioural tests showed that gravid females display a positive response towards the candidate-containing fraction, while males and non-gravid females showed no response. Our analyses demonstrate that this combined methodology is a promising approach for the discovery of peptides in amphibian chemical communication.

Une des périodes les plus critiques pour les plantes est l’établissement des jeunes plants. Cela est d’autant plus vrai pour les plantules de mangrove qui doivent, juste après leur abscission, gérer une haute salinité, un sol hypoxique, le courant engendré par les vagues et les marées. En plus des contraintes abiotiques, les propagules des mangroves sont souvent attaquées et mangées par des herbivores. Partie une: En Floride (USA), plusieurs insectes se nourrissent de Rhizophora mangle. L’insecte herbivore le plus représenté dans notre région d’étude est Coccotrypes rhizophorae, un Scolytinae d’environ 2 millimètres de long. Il infeste exclusivement les propagules et juvéniles de Rhizophora mangle, dans notre région d’étude, en creusant des galeries, en mangeant les tissues internes et en pondant ses œufs à l’intérieur. C. rhizophorae est connu pour être une menace à la régénération des mangroves. Cependant, R. mangle a un mécanisme de défense, les propagules peuvent réagir à l’attaque de ces insectes en développant des racines adventives juste au-dessus des tissues nécrosés par l’insecte. Nous avons focalisé notre étude sur ce phénomène. Plus spécifiquement, nous avons examiné si le développement de ces racines adventives au-dessus des dommages d’insectes peut offrir aux propagules infestées de meilleures chances de s’établir et de survivre (observations sur le terrain et sur plusieurs mois dans des conditions naturelles d’ombre et d’ensoleillement) (Chapitre 2). De plus, nous avons étudié comment le début de la croissance (contrôle sur un an dans des conditions artificielles sous serre) peut être impactée par les dommages d’insectes et par la présence de racines adventives (Chapitre 3). Les racines adventives induites par les dommages d’insectes peuvent remplacer les racines normales et rendre possible l’enracinement, même pour des propagules fortement endommagées par les insectes. Ces racines augmentent les chances d’établissement et de survie des propagules infestées. Cependant, quelques propagules infestées ont survécu et se sont établies sans développer de racines adventives mais ces propagules n’étaient que peu endommagées. De plus, le début de la croissance est affecté de manière différente selon l’intensité des dommages et la présence ou absence de racines adventives. Globalement, le taux de croissance des juvéniles était inversement proportionnel à la quantité de dommages. Cela peut être compensé par la présence de racines adventives, mais ce n’était pas toujours le cas. En effet, la compensation dépend de la partie attaquée. Les dommages localisés dans la partie supérieure de la propagule (près de la plumule) tendent à avoir un impact plus grand sur le début de la croissance. D’après nos résultats, nous pouvons conclure que les racines adventives induites par l’attaque peuvent remplacer les racines initiales et/ou normales. Dans certaines conditions, ces racines offrent, aux propagules infestées, la possibilité de survivre, de s’établir et de grandir de la même manière que les propagules non infestées. Dans ce contexte, nous pouvons affirmer que ces propagules sont tolérantes à l’herbivorie de cet insecte. Donc selon la disponibilité des propagules, leur habilité à être tolérante et le degré d’infestation de ces insectes, C. rhizophorae n’est peut-être pas une menace majeur pour la régénération de R. mangle.Partie deux: À Gazi Bay (Kenya), certaines familles de crabes herbivores (e.g. Sesarmidae, Gecarcinidae) sont connues pour consommer des propagules. Cette herbivorie peut affecter la régénération des mangroves dans des forêts naturelles et restaurées. L’herbivorie des crabes sur les propagules peut être affectée par de nombreux facteurs tant biotiques qu’abiotiques. Nous avons examiné comment certains de ces facteurs peuvent déterminer le comportement d’herbivorie de deux espèces de crabes (Neosarmatium africanum and Neosarmatium smithi) et comment ces facteurs peuvent stabiliser les interactions entre les herbivores et la végétation en répondant à cinq questions (Chapitre 4). Nous avons testé si :(1) la densité des crabes influence le taux d’herbivorie sur les propagules ;(2) la taille des crabes influence la compétition pour la nourriture et le taux d’herbivorie ;(3) la consommation des crabes est influencée par les espèces de propagules et de la couverture végétale ;(4) la densité de végétation est corrélée avec la densité de crabes ;et (5) les préférences alimentaires des crabes herbivores sont déterminées par la taille, la forme et la valeur nutritives des aliments. Nous avons trouvé que (1) le taux d’herbivorie sur les propagules est positivement corrélé à la densité de crabes. (2) La compétitivité des crabes n’est pas corrélée à leur taille. (3) Les propagules d’Avicennia marina sont plus rapidement déplacées que celles de Ceriops tagal sauf sous couvert de C. tagal. (4) La densité des crabes est négativement corrélée à la densité des arbres d’A. marina et de ses pneumatophores. (5) Les crabes préfèrent les aliments de petites tailles avec un ratio C:N faible. Nous avons trouvé qu’il y a une relation mutuelle entre la structure de la végétation et les populations de crabes. La compréhension de cette relation mutuelle peut être importante pour le succès et la gestion des forêts restaurées. Cette étude nous apprend comment les interactions mutuelles entre herbivores et propagules se stabilisent avec des mécanismes tels que la tolérance et la fuite ainsi que avec des facteurs intrinsèques et extrinsèques. Cependant, des études supplémentaires sont requises pour comprendre comment ces interactions entre herbivores et propagules peuvent évoluer avec les variations dues à la pression anthropique et aux changements climatiques., One of the most critical periods in a plant’s life cycle is seedling establishment. This is even more true for mangrove seedlings that immediately after abscission have to deal with high salinity, soil hypoxia, wave action and submergence by tides. Next to abiotic constraints, mangrove propagules are commonly attacked and consumed by herbivores (propagule predators). Part one: In Florida (USA), several insects feed on Rhizophora mangle. The most represented insect herbivore in our study region is Coccotrypes rhizophorae, a Scolytinae of about 2 millimeters long. It infests exclusively Rhizophora mangle propagules and juveniles, in our study region, by digging chambers, feeding on internal tissues and raising offspring inside. C. rhizophorae is known to be a threat to mangrove regeneration. Nevertheless, R. mangle has a mechanism of defence, propagules may react to insect attack by producing adventitious roots just above necrosed tissues. We focused our study on this phenomenon. More specifically, we examined whether development of induced roots above an attack could offer infested propagules better chances to establish and survive (several months in light and shade in natural field conditions) (Chapter 2). In addition, we investigated how early growth (one year under controlled greenhouse conditions) could be impacted by insect damage and presence of newly induced roots (Chapter 3). Induced roots could replace normal roots and make establishment possible, even for highly damaged propagules. They increased the chances of establishment and survival of infested propagules. However, some propagules that were attacked and only slightly damaged did not form induced roots but also survived and established. Moreover, early growth is affected differentially depending on damage intensity and presence or absence of induced roots. Globally, the juvenile growth rate was inversely proportional to the amount of damage. This could be compensated by presence of induced roots, but it was not always the case. Indeed, compensation depended on which part of the propagule was attacked. Damage located on the upper part of a propagule (towards the plumule) tended to have stronger impact on early growth. Following those results, we conclude that induced adventitious roots may replace initial and / or normal roots. In certain conditions they offer to infested propagules the ability to survive, establish and grow in a same way as non-infested propagules. In that context, we can confirm that those propagules are then tolerant to insect herbivory. Hence, depending on propagule availability, tolerance ability and degree of C. rhizophorae infestation, the insect may be not a major threat for R. mangle regeneration.Part two: Crabs play a major role in mangrove ecosystems. In Gazi Bay (Kenya), some herbivorous crab families (e.g. Sesarmidae, Gecarcinidae) are known to consume propagules. This herbivory can affect mangrove regeneration in natural and restored stands. Crab herbivory on propagules may be affected by many biotic and abiotic factors. We examined how some of the factors could determine the herbivory behavior of two cab species (Neosarmatium africanum and Neosarmatium smithi) and how those factors could stabilize herbivore-vegetation mutual interactions by answering five questions (Chapter 4). We tested whether: (1) crab density influences propagule herbivory rate; (2) crab size influences food competition and herbivory rate; (3) crabs depredate at different rates according to propagule and canopy cover species; (4) vegetation density is correlated with crab density; and (5) food preferences of herbivorous crabs are determined by size, shape and nutritional value. We found that (1) propagule herbivory rate was positively correlated to crab density. (2) Crab competition abilities were unrelated to their size. (3) Avicennia marina propagules were removed more quickly than Ceriops tagal except under C. tagal canopies. (4) Crab density was negatively correlated with the density of A. marina trees and pneumatophores. (5) Crabs prefer small items with a lower C:N ratio. There is a mutual relationship between stand characteristics and crab fauna, where stand composition and density influence predation and crab density, crab density impacts predation rates and crab size does not influence competition for mangrove propagules. Consequently, the mutual relationship between vegetation and crab populations seems to be important for forest restoration success and management. We conclude that this study gives answers on how herbivore-propagule mutual relationships are stabilized with tolerance, escape resistances and by intrinsic / extrinsic factors. However, more research is required to investigate how these herbivore-propagule interactions may evolve under increasing anthropic impacts, climate change and whether herbivore-propagule interactions are altered by these impacts and changes., Eén van de meest kritische periodes voor planten is de vestiging van de zaailingen. Dit is nog meer het geval voor zaailingen van mangroven, die meteen na abscissie kampen met hoge saliniteit, hypoxie in de bodem, golfenergie en getijden. Naast abiotische factoren, worden mangrovenpropagulen ook vaak aangevallen en opgegeten door herbivoren.Deel I: In Florida (VS) voeden verscheidene insecten zich met Rhizophora mangle. De meest voorkomende van de herbivore insecten in de bestudeerde regio is Coccotrypes rhizophorae, een ongeveer 2 millimeter lange Scolytinae. Deze tast bijna uitsluitend Rhizophora mangle propagulen en jonge plantjes aan, door gangen te graven in het weefsel, intern weefsel te eten en zich binnen de propagule voort te planten. C. rhizophorae staat bekend als een bedreiging voor de regeneratie van mangroven. Nochtans heeft R. mangle een tolerantie en verdedigingsmechanisme. Propagulen kunnen op de aanval van insecten reageren door net boven genecrotiseerd weefsel adventieve wortels te produceren. Onze studie richt zich op dit fenomeen. Meer specifiek hebben wij onderzocht of de ontwikkeling van geïnduceerde wortels boven een plaats op de propagule van een insectenaanval meer kans geeft aan aangetaste propagulen zich te vestigen en te overleven (waarneming gedurende meerdere maanden in licht en schaduw, natuurlijke veldomstandigheden) (Hoofdstuk 2). Tevens hebben wij bestudeerd hoe de vroege groei (één jaar onder gecontroleerde omstandigheden in een kas in België) beïnvloed kan worden door insectenschade en de aanwezigheid van recent geïnduceerde wortels (Hoofdstuk 3). Geïnduceerde wortels kunnen normale wortels vervangen en de vestiging mogelijk maken, zelfs voor zeer beschadigde propagulen. Dit vergrootte de kans op vestiging en overleving van de aangetaste propagulen. Sommige aangetaste propagulen die geen geïnduceerde wortels vormden, overleefden en vestigden zich daarentegen ook maar in dat geval waren ze slechts licht beschadigd. Bovendien wordt de vroege groei differentieel beïnvloed, afhankelijk van de intensiteit van de schade en van de aanwezigheid of afwezigheid van geïnduceerde wortels. Over het algemeen was de juveniele groeisnelheid omgekeerd evenredig met de hoeveelheid schade. Dit kon gecompenseerd worden door de aanwezigheid van geïnduceerde wortels, maar dat was niet altijd het geval. De compensatie hing af van het deel van de propagule dat aangevallen werd. Schade aan het bovenste deel van de propagule (bij de bladaanleg, pluimpje) heeft vaak meer impact op de vroege groei. Op basis van deze resultaten concluderen wij dat geïnduceerde incidentele wortels oorspronkelijke en/of normale wortels kunnen vervangen. In sommige omstandigheden geven ze aangetaste propagulen de mogelijkheid op dezelfde manier te overleven als niet-aangetaste propagulen, zich te vestigen en te groeien. In deze context kunnen we bevestigen dat deze propagulen tolerant zijn t.o. herbivore insecten. Daarom vormt het insect, afhankelijk van de beschikbaarheidsgraad van propagulen, van hun tolerantievermogen en van de infestatiegraad van C. rhizophorae, geen grote bedreiging voor de regeneratie van R. mangle in de onderzoek context van dit werk.Deel II: Krabben spelen een belangrijke rol in veel mangrove-ecosystemen. Voor het veel bestudeerde gebied in Gazi Bay (Kenia), weten we dat verscheidene families (e.g. Sesarmidae, Gecarcinidae) van herbivore of omnivore krabben zich voeden met propagulen. De herbivorie kan de mangroveregeneratie van natuurlijke en herstelde mangrovebestanden beïnvloeden. Deze krabbensoorten (die ook op propagulen foerageren) ondergaan zelf diverse invloeden van de in het systeem heersende biotische en abiotische factoren. Door vijf vragen te beantwoorden, bestudeerden wij hoe sommige van de factoren het herbivorie gedrag van twee krabbensoorten (Neosarmatium africanum en Neosarmatium smithi) kunnen bepalen en hoe die factoren de wederzijdse interactie tussen herbivoor en vegetatie kunnen stabiliseren (Hoofdstuk 4). Wij hebben getest of: (1) krabdensiteit de propagule herbivoorproportie beïnvloedt; (2) krabgrootte concurrentie en herbivoorproportie beïnvloedt; (3) schade door krabben op verschillende manieren gebeurt naargelang de verschillende propagulensoorten die gegeten worden en soorten die de boomlaag vormen; (4) vegetatiedensiteit gecorreleerd is met krabdensiteit; (5) voedingsvoorkeur van herbivore krabben bepaald wordt door grootte, vorm en nutritionele waarde (C:N ratio). We hebben geconstateerd dat (1) de proportie van herbivorie op propagulen positief gecorreleerd is met krabbendensiteit; (2) het competitief vermogen van krabben (voor herbivorie) geen verband heeft met hun grootte; (3) Avicennia marina propagulen sneller geconsumeerd worden dan die van Ceriops tagal, behalve onder bedekking van C. tagal zelf (boomlaag); (4) krabbendensiteit negatief gecorreleerd is met de densiteit van A. marina bomen en pneumatoforen (ademwortels); (5) krabben een voorkeur hebben voor kleine items met een lagere C:N ratio.Er bestaat een wederzijdse verhouding tussen groepskenmerken en de krabbenfauna. Waar de groepssamenstelling- en densiteit de predatie en krabbendensiteit beïnvloeden, heeft krabbendensiteit invloed op de intensiteit van predatie. Krabgrootte heeft geen impact op competitie voor mangrove propagulen. Bijgevolg zou de wederzijdse verhouding tussen vegetatie en krabbenpopulatie belangrijk kunnen zijn voor het succes en het beheer van bosrestauratie.Deze studie geeft inzicht in de wederzijdse verhouding tussen herbivoren en propagulen en hoe deze gestabiliseerd kan worden door tolerantie, ontsnappingsmechanismen, intrinsieke en extrinsieke factoren. Meer onderzoek is echter vereist om te bepalen hoe de interactie tussen herbivoren en propagulen zou kunnen evolueren met verandering als gevolg van menselijke druk en klimaatverandering, en of de aard en de uitkomst van de interacties zelf wijzigen t.g.v. de milieuverandering., Option Biologie des organismes du Doctorat en Sciences, info:eu-repo/semantics/nonPublished