Cloning, molecular characterization and tissue exPression of an octoPamine/tyramine recePtor from sPotted wing drosoPhila (DROSOPHILA SUZUKII)

Spotted wing Drosophila (Drosophila suzukii) is a polyphagous pest arrived in Europe in 2009 able to infest a growing number of fruit and vine species, causing considerable economic damage. D. suzukii grows very rapidly (seven to fifteen generations per year) and shows a remarkable ability to adapt to climatic conditions and to new host plants. These characteristics make its populations particularly difficult to control. Octopamine (OA) and tyramine (TA) biogenic amines are present in traces in vertebrates, while in invertebrates they act as substitutes for adrenaline and noradrenaline. Indeed, these amines regulate numerous physiological processes in insects. They exert their effects by binding to specific receptor proteins that belong to the superfamily of G-protein coupled receptors (GPCRs). In this work, we have isolated complementary DNA (cDNA) coding for an amine receptor from Drosophila suzukii (DsTyr). The cloned cDNA is about 1.8kb long and encodes for a 601 amino acids protein. This polypeptide presents the classical seven transmembrane domains as revealed by hydropathic profile analysis. BLAST analysis of the sequence shows a high identity (>98%) to the octopamine/tyramine receptor from Drosophila melanogaster. DsTyr1 deduced sequence will be compared to the amino acid sequence of octopamine/tyramine receptors from other insects. Furthermore, the various receptor sequences will be characterized by phylogenetic analysis. The expression level of the receptor will be studied by qRT-PCR analysis in different parts of D. suzukii male and female body (head, thorax and abdomen). With this work, we present a first structural and functional description of an octopamine/tyramine receptor from Drosophila suzukii

The Insect Type 1 Tyramine Receptors: From Structure to Behavior

Tyramine is a neuroactive compound that acts as neurotransmitter, neuromodulator, and neurohormone in insects. Three G protein-coupled receptors, TAR1-3, are responsible for mediating the intracellular pathway in the complex tyraminergic network. TAR1, the prominent player in this system, was initially classified as an octopamine receptor which can also be activated by tyramine, while it later appeared to be a true tyramine receptor. Even though TAR1 is currently considered as a well-defined tyramine receptor and several insect TAR1s have been characterized, a defined nomenclature is still inconsistent. In the last years, our knowledge on the structural, biochemical, and functional properties of TAR1 has substantially increased. This review summarizes the available information on TAR1 from different insect species in terms of basic structure, its regulation and signal transduction mechanisms, and its distribution and functions in the brain and the periphery. A special focus is given to the TAR1-mediated intracellular signaling pathways as well as to their physiological role in regulating behavioral traits. Therefore, this work aims to correlate, for the first time, the physiological relevance of TAR1 functions with the tyraminergic system in insects. In addition, pharmacological studies have shed light on compounds with insecticidal properties having TAR1 as a target and on the emerging trend in the development of novel strategies for pest control

Mating behaviour and dual mode communication of Pear Psylla CACOPSYLLA PYRI

Cacopsylla pyri (L.) (Hemiptera, Psyllidae) is one of the most important pests of European pear, and its management generally depends on the use of chemical insecticides, but C. pyri outbreaks are sometime observed. Ecological control strategies should be desirable and the knowledge of mating behavior is crucial to develop new ones. A multi-approaches research aimed to acquire knowledges about C. pyri mate finding. Electroantennographic (EAG) analyses and olfactometric bioassays were used to evaluate the activity of intraspecific semiochemicals on C. pyri. The EAG amplitudes revealed that volatile compounds, present in female cuticular extracts, elicited dose-dependent responses in males, indicating that these compounds were able to stimulate the male olfactory system. In behavioral bioassays, living females and female cuticular extracts, attracted summerform males in a highly significant manner. Gas chromatography-mass spectrometry revealed that 13-methylheptacosane, 11,13-dimethylheptacosane, 2-methylheptacosane and 3-methylheptacosane were found in larger amounts in female extracts than in male ones, which suggests their role in male attraction. In addition, a laser vibrometer device was used to detect a male-female substrate-born vibrations pattern during pre-copulatory period. The female vibrational signal was recorded as mp3 and conveyed, in loop using a minishaker, on pear shoots with C. pyri virgin pairs to interfere with the mating by masking the natural communications

Mining genes involved in indoxacarb resistance of Lobesia botrana (Denis and Schifferm\ufcller) by de novo transcriptome assembly and differential expression analysis.

Lobesia botrana (Denis and Schifferm\ufcller) (Lepidoptera: Tortricidae) is one of the most important grapevine pests in Europe but, being a non-model organism, only limited genomic and transcriptomic resources are available for functional studies at the molecular level, such as those relevant to insecticide resistance and pest control. Hence, to gain insight into the mechanism of indoxacarb resistance, a blocker of insect voltage-gated sodium channels (NaV), we analysed the transcriptome and expression profile in 2nd instars of L. botrana from susceptible and field selected populations (LC50 resistance ratio 72). De novo transcriptome assembly using Trinity resulted in 141,581 isoforms clustered in 94,290 putative genes. The transcriptome completeness was supported by BUSCO: 92% of conserved orthologs (n= 1,658) were retrieved as a complete sequence, 6.3% displayed fragmented ORFs, and only 1.7% were missing. 36,250 genes were preliminary annotated relaying on the longest isoform per gene, by running Annocript pipeline against non-redundant protein databases (Nr), gene ontology (GO), cluster of orthologous groups of proteins (COG), KEGG orthology (KO) and long non-coding RNAs (lncRNAs). Conditional Reciprocal Best BLAST analysis of protein isoforms performed on Lepidoptera proteomes identified putative orthologs of multigene family members potentially involved in metabolic resistance (61 cytochrome P450 monooxygenases, 25 glutathione S-transferases, 13 carboxylesterases, 25 UDP-glucuronosyltransferases) as well as alternatively spliced isoforms of the NaV gene. Among 263 upregulated and annotated genes in the resistant population, functional GO enrichment analysis revealed overrepresentation of terms for cytochrome P450, due to up-regulation of CYP6B and CYP9A subfamily members as well as increased transcript level for UGT genes. Hydrolases were, on the contrary, overrepresented in 293 annotated genes, downregulated in the resistant population. These data tentatively suggest the reduced susceptibility to indoxacarb might be related to an increase of Phase I and II detoxification along with reduced bioactivation of the insecticide

Spectrometer for X-ray emission experiments at FERMI free-electron-laser

A portable and compact photon spectrometer to be used for photon in-photon out experiments, in particular x-ray emission spectroscopy, is presented. The instrument operates in the 25\u2013800 eV energy range to cover the full emissions of the FEL1 and FEL2 stages of FERMI. The optical design consists of two interchangeable spherical varied-lined-spaced gratings and a CCD detector. Different input sections can be accommodated, with/without an entrance slit and with/without an additional relay mirror, that allow to mount the spectrometer in different end-stations and at variable distances from the target area both at synchrotron and at free-electron-laser beamlines. The characterization on the Gas Phase beamline at ELETTRA Synchrotron (Italy) is presented

Three-Dimensional Shapes of Spinning Helium Nanodroplets

A significant fraction of superfluid helium nanodroplets produced in a free-jet expansion have been observed to gain high angular momentum resulting in large centrifugal deformation. We measured single-shot diffraction patterns of individual rotating helium nanodroplets up to large scattering angles using intense extreme ultraviolet light pulses from the FERMI free-electron laser. Distinct asymmetric features in the wide-angle diffraction patterns enable the unique and systematic identification of the three-dimensional droplet shapes. The analysis of a large dataset allows us to follow the evolution from axisymmetric oblate to triaxial prolate and two-lobed droplets. We find that the shapes of spinning superfluid helium droplets exhibit the same stages as classical rotating droplets while the previously reported metastable, oblate shapes of quantum droplets are not observed. Our three-dimensional analysis represents a valuable landmark for clarifying the interrelation between morphology and superfluidity on the nanometer scale

Attosecond pulse shaping using a seeded free-electron laser

Attosecond pulses are central to the investigation of valence- and core-electron dynamics on their natural timescales1–3. The reproducible generation and characterization of attosecond waveforms has been demonstrated so far only through the process of high-order harmonic generation4–7. Several methods for shaping attosecond waveforms have been proposed, including the use of metallic filters8,9, multilayer mirrors10 and manipulation of the driving field11. However, none of these approaches allows the flexible manipulation of the temporal characteristics of the attosecond waveforms, and they suffer from the low conversion efficiency of the high-order harmonic generation process. Free-electron lasers, by contrast, deliver femtosecond, extreme-ultraviolet and X-ray pulses with energies ranging from tens of microjoules to a few millijoules12,13. Recent experiments have shown that they can generate subfemtosecond spikes, but with temporal characteristics that change shot-to-shot14–16. Here we report reproducible generation of high-energy (microjoule level) attosecond waveforms using a seeded free-electron laser17. We demonstrate amplitude and phase manipulation of the harmonic components of an attosecond pulse train in combination with an approach for its temporal reconstruction. The results presented here open the way to performing attosecond time-resolved experiments with free-electron lasers

Complex attosecond waveform synthesis at fel fermi

Free-electron lasers (FELs) can produce radiation in the short wavelength range extending from the extreme ultraviolet (XUV) to the X-rays with a few to a few tens of femtoseconds pulse duration. These facilities have enabled significant breakthroughs in the field of atomic, molecular, and optical physics, implementing different schemes based on two-color photoionization mechanisms. In this article, we present the generation of attosecond pulse trains (APTs) at the seeded FEL FERMI using the beating of multiple phase-locked harmonics. We demonstrate the complex attosecond waveform shaping of the generated APTs, exploiting the ability to manipulate independently the amplitudes and the phases of the harmonics. The described generalized attosecond waveform synthesis technique with an arbitrary number of phase-locked harmonics will allow the generation of sub-100 as pulses with programmable electric fields

Tyraminergic signaling in phytophagous insects: from physiology to pest control

Le ammine biogene tiramina (TA) ed octopamina (OA) sono sostanze neurochimiche, essenziali negli invertebrati, che agiscono come sostituti dell'adrenalina e della noradrenalina. TA ed OA esercitano i loro effetti legando specifici recettori che appartengono alla superfamiglia dei recettori accoppiati a proteine G. I recettori tiramici (TAR) ed octopaminici (OAR) svolgono un ruolo importante nel modulare la biologia, la fisiologia e il comportamento degli insetti. In questa tesi di dottorato viene descritta la caratterizzazione del recettore della tiramina di tipo 1 (TAR1) in due insetti nocivi per le piante, Drosophila suzukii e Halyomorpha halys, con lo scopo di investigare il ruolo che il recettore esercita nel controllo dei tratti fisiologici e comportamentali oltre che testare TAR1 come possibile bersaglio per biopesticidi. Il TAR1 di D. suzukii si è rivelato un interessante bersaglio per biopesticidi, nello specifico i monoterpeni. Questi composti sono stati in grado di modulare quegli aspetti fisiologici e comportamentali di D. suzukii direttamente controllati da TAR1. In H. halys, la downregolazione di TAR1 attraverso RNAi ha evidenziato un possibile coinvolgimento del recettore nella percezione dei feromoni. Insieme, i dati presentati mostrano come TAR1 sia cruciale nel controllo di numerosi aspetti fisiologici e comportamentali degli insetti. Inoltre, questo recettore sembra essere un bersaglio per approcci innovativi e rispettosi dell'ambiente nel controllo dei parassiti.The biogenic amines tyramine (TA) and octopamine (OA) are neurochemicals essential in invertebrates that act as adrenaline and noradrenaline substitutes. They exert their effects by binding specific receptor proteins that belong to the superfamily of G-protein coupled receptors. Tyramine (TAR) and octopamine (OAR) receptors play important roles in modulating the biology, physiology and behavior of insect. This PhD thesis describes the characterization of the type 1 tyramine receptor (TAR1) in two insect pests, Drosophila suzukii and Halyomorpha halys, to dissect the receptor role in controlling physiological and behavioral traits as well as to examine TAR1 as possible target for biopesticides. D. suzukii TAR1 proved to be an interesting target for biopesticides, such as monoterpenes. These compounds were in fact able to modulate directly TAR1-controlled physiology and behavior. In H. halys, RNAi-mediated TAR1 downregulation suggested that the receptor involvement in pheromone perception. Together, the data described emphasize TAR1 as crucial in controlling and defining physiological and behavioral aspects in insects. Furthermore, this receptor appears an interesting target for innovative and environmental friendly in pest control

Monoterpenes-induced toxicity in nymphal stages of Halyomorpha halys

In this work, the toxic activity by fumigation of 10 monoterpenes was evaluated on Halyomorpha halys 2nd instar nymphs. The results showed that (±)-limonene was the monoterpene with the highest insecticidal activity (LC50 0.48 µl l−1 air), followed by two phenolic monoterpenes eugenol and isoeugenol (LC50 0.41 and 0.55 µl l−1 air, respectively). In addition, the alcoholic monoterpenes α-terpineol and l -(−)-menthol and the ketonic monoterpene (+)-pulegone exhibited strong efficacy on H. halys 2nd instar nymphs (LC50 values 0.84, 1.15 and 0.48 µl l−1 air, respectively). These preliminary data indicate that essential oils and the individual terpenic components could be used as fumigants for the control of H. halys nymphs