Post-mating inhibition of sex pheromone responses in a male moth: search for a brain and/or sex gland factor

Viele Tiere benutzen ihren Geruchssinn zum Auffinden von Nahrung und Geschlechtspartnern. Bei vielen Nachtfaltern ist dazu ein besonders effektives System der Partnerfindung evolviert, bei dem das Weibchen Sexualpheromone produziert und aussendet, die von den Männchen besonders gut wahrgenommen werden können. Die Sensibilität der Männchen für die arteigenen Sexualpheromone ist so hoch, dass eine Partnerfindung über lange Distanzen möglich ist. Die männliche Ypsiloneule, Agrotis ipsilon, erlebt jedoch nach der Paarung eine Periode von verringerter Sensibilität auf das artspezifische Sexualpheromon, die Post-Ejakulations Intervall (PEI) genannt wird. Während dieser Phase werden die männlichen Falter nicht von Sexualpheromon bzw. den pheromonaussendenden Weibchen angezogen. Das PEI dauert bis zur nächsten nächtlichen Aktivitätsperiode, von diesem Zeitpunkt an ist die ursprüngliche Sensibilität vollständig wiederhergestellt. Dieses Pausieren des Sexualverhaltens erlaubt den verpaarten Männchen für die Fortpflanzung wichtige Proteine in den Akzessorischen Sexualdrüsen (ASD) zu sammeln, die bei der vorherigen Paarung vollständig entleert wurden. Damit verhindern die Männchen Verpaarungen mit geringer Befruchtungswahrscheinlichkeit. Diese Plastizität im Sexualverhalten wird maßgeblich bestimmt durch eine verringerte Sensibilität auf Sexualpheromon der Projektionsneurone (PN) in den primären olfaktorischen Zentren, den Antennalloben (AL). Das beobachtete PEI könnte durch Veränderungen in der Produktion neuroregulatorischer Peptide, biogener Amine oder Ecdysteroidhormone reguliert werden. Auch wenn bis dato keine Mechanismen bekannt sind, die das PEI bei Insektenmännchen auslösen, so ist doch bei Weibchen vieler Insektenarten (zum Beispiel aus den Ordnungen Diptera oder Lepidoptera) eine Refraktärperiode bekannt, die durch eine vorhergegangene Paarung ausgelöst wird. Dabei werden vom Männchen Peptide wie das Sex-Peptid (SP) übertragen, die von den männlichen ASD gebildet werden und im weiblichen Organismus über spezifische Rezeptoren wirken. Es ist möglich, dass im männlichen Organismus ähnliche Peptide das PEI auslösen. Um das Ursprungsorgan des männlichen PEI zu finden, wurden die Effekte von Injektionen mit Gehirnen oder ASD auf das Verhalten von unverpaarten und verpaarten männlichen Faltern in Windtunnelexperimenten beobachtet. Wesentlich wurden zwei Hypothesen verfolgt: i) unverpaarte Männchen produzieren eine Substanz die ihre Empfindlichkeit für das weibliche Pheromon aufrechterhält, und die nach der Paarung nicht mehr oder in verringertem Maß produziert wird & ii) verpaarte Männchen erzeugen auf Grund der Paarung eine Substanz die das PEI induziert. Zusätzlich wurden die möglicherweise unterschiedlichen Peptidprofile in den Gehirnen unverpaarter und verpaarter Männchen mittels MALDI-TOF Massenspektrometrie verglichen. Die Ergebnisse zeigen deutlich, dass das Verhalten verpaarter Männchen nicht durch die Injektion von Geweben unverpaarter Männchen beeinflussbar ist. Im Gegensatz dazu ist es durchaus möglich mittels der Injektion von ASD, aber nicht Gehirnen, verpaarter Männchen eine Reduktion des Verhaltens unverpaarter Männchen zu erreichen. Weiters zeigen die vorläufigen Ergebnisse der MALDI-TOF Massenspektrometrie keine drastischen Veränderungen im Peptideprofil nach der Paarung. Demnach kann geschlossen werden dass die männlichen ASD Substanzen produzieren, die nicht nur das Verhalten des Weibchens verändern, sondern auch auf das Männchen selbst einwirken. Nachdem verpaarte Männchen Verhaltensänderungen zeigen die denen verpaarter Weibchen ähneln, wird angenommen, dass ein Peptid ähnlich dem SP das männliche PEI auslöst. Alternativ dazu könnte die Steuerung des PEI durch Dopamin (DA) oder das Insektenhormon 20-Hydroxyecdyson erfolden, da beide Substanzen nachgewiesenermaßen von großer Bedeutung für die männliche Sexualität von Vertebraten und Insekten sind.Olfaction is used by most animals to find adequate food sources and sexual partners. Newly-mated males of the moth Agrotis ipsilon experience a post-ejaculation interval (PEI), during which they are not attracted to the female-produced sex pheromone. This period lasts until the next night when the sensitivity is fully restored, and allows the males to refill their reproductive accessory sex glands for a potential new mating. This behavioural plasticity is accompanied by a reduced sensitivity of projection neurons (PN) in the primary olfactory centre, the antennal lobes (AL). This observed PEI could be due to a down- or up-regulation of neuroregulatory peptides, biogenic amines or ecdysteroid hormones. Whereas there is no mechanism known to trigger PEI in insect males, a period of non-receptivity following mating was demonstrated in females of many insect species (e.g. Diptera, Lepidoptera). The female reproductive refractory phase is induced by proteins such as the sex peptide (SP) derived from the male accessory sex glands (ASGs), which are transferred during the copulation and act through the neuronal pathway via specific receptors. It might be possible that similar factors could have comparable effects in males. In order to elucidate the origin of the observed PEI in A. ipsilon males, the effects of injections of brains or ASGs on the physiology of virgin and mated males were examined by windtunnel experiments. Two main hypotheses were tested i) virgin males express in their tissues a substance that renders them sensitive to the female pheromone, which is no longer produced after mating; ii) mated males produce a substance as a result of mating that induces the PEI. Additionally, putatively differing peptide profiles of brains of virgin and mated males were compared by means of MALDI-TOF. The results show that the behaviour of mated males could not be restored by the injection of tissues from virgin males. Contrarily, injections of ASGs, but not brains, of mated males significantly reduced the behavioural response of virgin males. Lastly, preliminary MALDI-TOF results show that the peptide profile is not drastically changed after mating. Thus it is concluded that the male ASGs produce substances that not only affect the female’s behaviour, but also act on the male itself. As males show changes in their behaviour similar to unreceptive females, it is presumed that a SP-like peptide induces the male PEI. Alternatively, the PEI could be triggered by dopamine (DA) or the insect horomone 20-Hydroxyecdysone (20-OH-E), as these substances have been shown to be of great importance in male sexuality in both vertebrates and insects

Assessing the Ecological Status of European Rivers and Lakes Using Benthic Invertebrate Communities: A Practical Catalogue of Metrics and Methods

The Water Framework Directive requires that the ecological status of surface waters be monitored and managed if necessary. A central function in ecological status assessment has the Biological Quality Elements-organisms inhabiting surface waters-by indicating human impact on their habitat. For benthic invertebrates, a wide array of national methods are used, but to date no comprehensive summary of metrics and methods is available. In this study, we summarize the benthic invertebrate community metrics used in national systems to assess the ecological status of rivers, (very) large rivers, and lakes. Currently, benthic invertebrate assemblages are used in 26 national assessment systems for rivers, 13 assessment systems for very large rivers, and 21 assessment systems for lakes in the EU. In the majority of systems, the same metrics and modules are used. In the Red Queen's race of ecosystem management this may be a disadvantage as these same metrics and module likely depict the same stressors but there is growing evidence that aquatic ecosystems are subject to highly differentiated, complex multiple stressor impacts. Method development should be fostered to identify and rank impacts in multi-stressor environments. DNA-based biomonitoring 2.0 offers to detect stressors with greater accuracy-if new tools are calibrated

Molecular association and morphological characterisation of Himalopsyche larval types (Trichoptera, Rhyacophilidae)

Himalopsyche Banks, 1940 (Trichoptera, Rhyacophilidae) is a genus of caddisflies inhabiting mountain and alpine environments in Central and East Asia and the Nearctic. Of 53 known species, only five species have been described previously in the aquatic larval stage. We perform life stage association using three strategies (GMYC, PTP, and reciprocal monophyly) based on fragments of two molecular markers: the nuclear CAD, and the mitochondrial COI gene. A total of 525 individuals from across the range of Himalopsyche (Himalayas, Hengduan Shan, Tian Shan, South East Asia, Japan, and western North America) was analysed and 32 operational taxonomic units (OTUs) in our dataset delimited. Four distinct larval types of Himalopsyche are uncovered, and these are defined as the phryganea type, japonica type, tibetana type, and gigantea type and a comparative morphological characterisation of the larval types is presented. The larval types differ in a number of traits, most prominently in their gill configuration, as well as in other features such as setal configuration of the pronotum and presence/absence of accessory hooks of the anal prolegs

The last hideout: Abundance patterns of the not-quite-yet extinct mayfly Prosopistoma pennigerum in the Albanian Vjosa River network

1. The mayfly Prosopistoma pennigerum (Müller, 1785) (Insecta: Ephemeroptera) once occurred in many European river networks. However, observations decreased in the last decades and the species can be considered largely extinct throughout Europe due to river alterations. 2. Only three extant populations are known from Cabriel (southern Spain), Volga (Russia) and Vjosa (Albania) rivers. 3. We recorded the species along a 150 km stretch in the Vjosa River in three sampling seasons (spring 2018, fall 2018 and fall 2019), counting up to 302 P. pennigerum per m2, the highest recorded abundance for the species to date. Moreover, we detected traces of environmental DNA in a newly designed targeted eDNA assay. 4. In our modelling approach we define the species’ niche in a theoretically available niche space given by the Vjosa River network and predict a high probability of presence (θ) in downstream located sections of this river. Expected abundances (λ) could be related to a set of environmental variables, importantly to higher discharge and increased sediment dynamics. 5. Simultaneous occurrence of larvae of different sizes at individual sites suggests an asynchronous life cycle, which may be advantageous to cope with the highly dynamic river hydrology. 6. The P. pennigerum population in the Vjosa is of key importance for the species’ global survival

The Vjosa River corridor: a model of natural hydro-morphodynamics and a hotspot of highly threatened ecosystems of European significance

Context: Large near-natural rivers have become rare in Europe, a fact reflected in the high conservation status of many riverine ecosystems. While the Balkan still harbors several intact river corridors, most of these are under pressure from planned hydropower constructions. Unfortunately, there is little information available on the hydromorphodynamics and biota of Balkan rivers under threat. Objectives: We present a synthesis of research on the Vjosa in Southern Albania. Here, longitudinal continuity in water flow, undisturbed sediment transport and intact fluvial dynamics are still maintained, but threatened by two large dams planned in its downstream section. We intend to provide a first multidisciplinary inventory of this river system as an example of the knowledge base required for sound water management decisions in the Balkans. Methods: Based on field work of a multidisciplinary consortium of scientists from Albania and other countries conducted from 2017 onwards, we summarize the most important findings on geomorphology of the riverine landscape, habitat turnover rates, vegetation ecology and selected animal taxa. Results: We found evidence that significant areas (86%) of the river corridor are covered by habitats listed in Annex 1 of the European Union Habitats Directive. These are associated with a high number of threatened biota. Conclusions: Our findings underscore the value of the Vjosa as one of the few remaining reference sites for dynamic floodplains in Europe and as a natural laboratory for interdisciplinary research. We emphasize that such multidisciplinary studies are a prerequisite for informed evaluation of potential impacts caused by hydropower plants

Gill Structure Linked to Ecological and Species Diversification in a Clade of Caddisflies

Streams represent a special case of directional environmental gradients where ecological opportunity for diversification may be associated with upstream and downstream dispersal into habitats that differ in selective pressures. Temperature, current velocity and variability, sediment erosion dynamics and oxygen saturation are key environmental parameters that change in predictable ways from springs to river mouth. Many aquatic insects occupy specific longitudinal regions along these gradients, indicating a high degree of adaptation to these specific environmental conditions. In caddisflies, the evolution of tracheal gills in larval and pupal stages may be a major driver in oxygen uptake efficiency and ecological diversification. Here we study the evolution of larval gill structure in the Rhyacophila vulgaris species group using phylogenomic methods. Based on anchored hybrid enrichment, we sequenced 97 kbp of data representing 159 independent nuclear protein coding gene regions to infer the phylogeny of the R. vulgaris species group, whose species exhibit both high diversity of gill types and varied longitudinal preferences. We find that the different gill types evolved independently as derived characters in the genus and that gill structure is linked to the longitudinal habitat preference, thereby serving as a possible ecological key innovation in the R. vulgaris group

Why we need sustainable networks bridging countries, disciplines, cultures and generations for Aquatic Biomonitoring 2.0: A Perspective Derived From the DNAqua-Net COST Action

Aquatic biomonitoring has become an essential task in Europe and many other regions as a consequence of strong anthropogenic pressures affecting the health of lakes, rivers, oceans and groundwater. A typical assessment of the environmental quality status, such as it is required by European but also North American and other legislation, relies on matching the composition of assemblages of organisms identified using morphological criteria present in aquatic ecosystems to those expected in the absence of anthropogenic pressures. Through decade-long and difficult intercalibration exercises among networks of regulators and scientists in European countries, a pragmatic biomonitoring approach was developed and adopted, which now produces invaluable information. Nonetheless, this approach is based on several hundred different protocols, making it susceptible to issues with comparability, scale and resolution. Furthermore, data acquisition is often slow due to a lack of taxonomic experts for many taxa and regions and time-consuming morphological identification of organisms. High-throughput genetic screening methods such as (e)DNA metabarcoding have been proposed as a possible solution to these shortcomings. Such "next-generation biomonitoring", also termed "biomonitoring 2.0", has many advantages over the traditional approach in terms of speed, comparability and costs. It also creates the potential to include new bioindicators and thereby further improves the assessment of aquatic ecosystem health. However, several major conceptual and technological challenges still hinder its implementation into legal and regulatory frameworks. Academic scientists sometimes tend to overlook legal or socioeconomic constraints, which regulators have to consider on a regular basis. Moreover, quantification of species abundance or biomass remains a significant bottleneck to releasing the full potential of these approaches. Here, we highlight the main challenges for next-generation aquatic biomonitoring and outline principles and good practicCOST - European Cooperation in Science and Technology(CA15219). COST Action DNAqua-Net (CA15219), supported by the COST (European Cooperation in Science and Technology) programm

Differential Interactions of Sex Pheromone and Plant Odour in the Olfactory Pathway of a Male Moth

Most animals rely on olfaction to find sexual partners, food or a habitat. The olfactory system faces the challenge of extracting meaningful information from a noisy odorous environment. In most moth species, males respond to sex pheromone emitted by females in an environment with abundant plant volatiles. Plant odours could either facilitate the localization of females (females calling on host plants), mask the female pheromone or they could be neutral without any effect on the pheromone. Here we studied how mixtures of a behaviourally-attractive floral odour, heptanal, and the sex pheromone are encoded at different levels of the olfactory pathway in males of the noctuid moth Agrotis ipsilon. In addition, we asked how interactions between the two odorants change as a function of the males' mating status. We investigated mixture detection in both the pheromone-specific and in the general odorant pathway. We used a) recordings from individual sensilla to study responses of olfactory receptor neurons, b) in vivo calcium imaging with a bath-applied dye to characterize the global input response in the primary olfactory centre, the antennal lobe and c) intracellular recordings of antennal lobe output neurons, projection neurons, in virgin and newly-mated males. Our results show that heptanal reduces pheromone sensitivity at the peripheral and central olfactory level independently of the mating status. Contrarily, heptanal-responding olfactory receptor neurons are not influenced by pheromone in a mixture, although some post-mating modulation occurs at the input of the sexually isomorphic ordinary glomeruli, where general odours are processed within the antennal lobe. The results are discussed in the context of mate localization

The future of biotic indices in the ecogenomic era: Integrating (e)DNA metabarcoding in biological assessment of aquatic ecosystems

The bioassessment of aquatic ecosystems is currently based on various biotic indices that use the occurrence and/or abundance of selected taxonomic groups to define ecological status. These conventional indices have some limitations, often related to difficulties in morphological identification of bioindicator taxa. Recent development of DNA barcoding and metabarcoding could potentially alleviate some of these limitations, by using DNA sequences instead of morphology to identify organisms and to characterize a given ecosystem. In this paper, we review the structure of conventional biotic indices, and we present the results of pilot metabarcoding studies using environmental DNA to infer biotic indices. We discuss the main advantages and pitfalls of metabarcoding approaches to assess parameters such as richness, abundance, taxonomic composition and species ecological values, to be used for calculation of biotic indices. We present some future developments to fully exploit the potential of metabarcoding data and improve the accuracy and precision of their analysis. We also propose some recommendations for the future integration of DNA metabarcoding to routine biomonitoring programs.info:eu-repo/semantics/publishedVersio

Assessing the Ecological Status of European Rivers and Lakes Using Benthic Invertebrate Communities: A Practical Catalogue of Metrics and Methods

The Water Framework Directive requires that the ecological status of surface waters be monitored and managed if necessary. A central function in ecological status assessment has the Biological Quality Elements—organisms inhabiting surface waters—by indicating human impact on their habitat. For benthic invertebrates, a wide array of national methods are used, but to date no comprehensive summary of metrics and methods is available. In this study, we summarize the benthic invertebrate community metrics used in national systems to assess the ecological status of rivers, (very) large rivers, and lakes. Currently, benthic invertebrate assemblages are used in 26 national assessment systems for rivers, 13 assessment systems for very large rivers, and 21 assessment systems for lakes in the EU. In the majority of systems, the same metrics and modules are used. In the Red Queen’s race of ecosystem management this may be a disadvantage as these same metrics and module likely depict the same stressors but there is growing evidence that aquatic ecosystems are subject to highly differentiated, complex multiple stressor impacts. Method development should be fostered to identify and rank impacts in multi-stressor environments. DNA-based biomonitoring 2.0 offers to detect stressors with greater accuracy—if new tools are calibrated