Multiple Changes in Peptide and Lipid Expression Associated with Regeneration in the Nervous System of the Medicinal Leech

peer reviewe

Design and planning of a transdisciplinary investigation into farmland pollinators: rationale, co-design, and lessons learned

To provide a complete portrayal of the multiple factors negatively impacting insects in agricultural landscapes it is necessary to assess the concurrent incidence, magnitude, and interactions among multiple stressors over substantial biogeographical scales. Trans-national ecological field investigations with wide-ranging stakeholders typically encounter numerous challenges during the design planning stages, not least that the scientific soundness of a spatially replicated study design must account for the substantial geographic and climatic variation among distant sites. ‘PoshBee’ (Pan-European assessment, monitoring, and mitigation of Stressors on the Health of Bees) is a multi-partner transdisciplinary agroecological project established to investigate the suite of stressors typically encountered by pollinating insects in European agricultural landscapes. To do this, PoshBee established a network of 128 study sites across eight European countries and collected over 50 measurements and samples relating to the nutritional, toxicological, pathogenic, and landscape components of the bees’ environment. This paper describes the development process, rationale, and end-result of each aspect of the of the PoshBee field investigation. We describe the main issues and challenges encountered during the design stages and highlight a number of actions or processes that may benefit other multi-partner research consortia planning similar large-scale studies. It was soon identified that in a multi-component study design process, the development of interaction and communication networks involving all collaborators and stakeholders requires considerable time and resources. It was also necessary at each planning stage to be mindful of the needs and objectives of all stakeholders and partners, and further challenges inevitably arose when practical limitations, such as time restrictions and labour constraints, were superimposed upon prototype study designs. To promote clarity for all stakeholders, for each sub-component of the study, there should be a clear record of the rationale and reasoning that outlines how the final design transpired, what compromises were made, and how the requirements of different stakeholders were accomplished. Ultimately, multi-national agroecological field studies such as PoshBee benefit greatly from the involvement of diverse stakeholders and partners, ranging from field ecologists, project managers, policy legislators, mathematical modelers, and farmer organisations. While the execution of the study highlighted the advantages and benefits of large-scale transdisciplinary projects, the long planning period emphasized the need to formally describe a design framework that could facilitate the design process of future multi-partner collaborations

Lipidomic studies and developments in imaging mass spectrometry of lipids : application to the regeneration of Hirudo medicinalis central nervous system

Les lipides constituent un ensemble de métabolites se déclinant en plusieurs classes. Outre leur rôle structural, ils peuvent aussi agir dans différents mécanismes biologiques en tant que messagers cellulaires. Des recherches en lipidomique sur les facultés de régénération du système nerveux central (SNC) d’Hirudo medicinalis après une lésion ont été entreprises. Des images par spectrométrie de masse de type Tof-SIMS (Time-of-fligh Secondary Ion Mass spectrometry) réalisées sur le système nerveux lésé d’Hirudo ont démontré au cours du temps une apparition localisée d’acides gras et de triglycérides au niveau des neurones lésés. Une approche par spectrométrie de masse MALDI Tof-Tof (Matrix-Assisted Laser Desorption Ionisation Time-of-fligh) sur des extraits lipidiques de chaînes nerveuses en régénération ont démontré la présence d’une forte modulation de messagers secondaires lipidiques: le 2 arachidonyl glycerol (2AG) et l'anandamide (AEA) régulant l’activité sécrétoire du monoxyde d’azote (NO) par les cellules microgliales du SNC. A l’inverse des vertébrés, Hirudo est capable de réguler très finement sa libération de NO à l’endroit lésé du SNC ce qui favorise les mécanismes de réparation cellulaire après lésion. En parallèle, une nouvelle approche de développement de l'imagerie par spectrométrie de masse MALDI Tof-Tof des lipides a permis pour la première fois, de valider l'utilisation de colorants histologiques lipophiles en vue de localiser et de caractériser des lipides in situ sur coupes de cerveaux de rat. Cette nouvelle approche par les colorants qualifiée de DALDI Tof-Tof (Dye-Assisted Laser Desorption Ionisation Time-of-fligh) a trouvé des applications directes dans le cancer de l'ovaire où une expression différentielle de lipides a été démontrée entre les régions saine et cancéreuse. Ce travail a donc permis d'une part de mettre en évidence une forte, implication des lipides de type cannabinoïdes sur le recrutement des cellules microgliales ainsi qu'une régulation de la production du NO au site de la lésion, et d'autre part, l'apparition de lipides spécifiques au sein des neurones au cours des phases de régénération nerveuse du SNC d'Hirudo. De plus, l'utilisation de colorants histologiques comme matrice en vue de détecter des lipides directement sur coupes de tissus permet d'associer l'application de l'imagerie MALDI des lipides à la recherche de marqueurs potentiels de nature lipidique dans le cancer de l'ovaire. Des perspectives d'interaction avec la biologie clinique et médicale pourraient être envisagées afin de compléter les diagnostics des anatomo-pathologistes par une localisation et une caractérisation des lipides impliqués dans une pathologie donnéeLipids are metabolites which can be declined in many classes. Most of them are known to figure as structural molecules in the cell membrane but a significant key role is also played as cellular messenger when interacting with specific membrane receptors. The leech Hirudo medicinalis which is a model for the neurosciences is able to regenerate its central nervous system (CNS) after a lesion in opposition with the CNS of vertebrate. In this way, we showed by Tof-SIMS imaging changes in the lipidome of harmed leech brain directly on tissue. In particular lipids such as fatty acids and triglycerides have been characterized during injuries of the leech CNS. These lipids were expressed in specific areas of damaged neurons. We also characterized endocannabinoids in crude lipid extracts of harmed leech brain by MALDI-Tof Tof mass spectrometry. The identified endocannabinoids anandamide (AEA) and 2-arachidonyl glycerol (2AG) are lipids which act as cellular messenger. During the injury of the leech brain, an increase in the 2AG concentration in the brain were demonstrated while AEA was decreasing. As a consequence, a modulation in the chemotactic effect on microglial cells was observed in the harmed connective. 2AG and AEA have also been demonstrated to regulate the NO release in activated microglia under neurotoxic rate. A new approach in developpemental research in lipidomic using MALDI imaging mass spectrometry allowed us to validate for the first time the use of specific histochemical dies for lipids as a way to detect and characterize lipids directly on rat brain tissue. This new approach of studiying lipids using dies named DALDI Tof-Tof (Dye-Assisted Laser Desorption Ionisation Time-of-fligh)has been successfully applied to the ovarian cancer where different patterns in the lipid content were detected on tissue slices between benign and malignant areas. This work highlighted in one hand the roles played by cannabinoids on the microglia chemotaxis and the regulation of NO secretion at the lesion site and in the second hand, the expression of specific lipids on neurons close to the lesion site during the regeneration process of the damaged leech CNS. Moreover, by using histological dies specific the the lipid staining as matrices, we showed that MALDI imaging of lipids allowed to detect lipids which could be potentially biomarkers for ovarian cancer. A cross-talk with scientist and medical fields can emerged since using both the same way to detect lipids on tissue for diagnosis purposes

Étude lipidomique et développements en imagerie MALDI des lipides (application à la régénération du système nerveux central d'Hirudo medicinalis)

Les lipides constituent un ensemble de métabolites se déclinant en plusieurs classes. Outre leur rôle structural, ils peuvent aussi agir dans différents mécanismes biologiques en tant que messagers cellulaires. Des recherches en lipidomique sur les facultés de régénération du système nerveux central (SNC) d Hirudo medicinalis après une lésion ont été entreprises. Des images par spectrométrie de masse de type Tof-SIMS (Time-of-fligh Secondary Ion Mass spectrometry) réalisées sur le système nerveux lésé d Hirudo ont démontré au cours du temps une apparition localisée d acides gras et de triglycérides au niveau des neurones lésés. Une approche par spectrométrie de masse MALDI Tof-Tof (Matrix-Assisted Laser Desorption Ionisation Time-of-fligh) sur des extraits lipidiques de chaînes nerveuses en régénération ont démontré la présence d une forte modulation de messagers secondaires lipidiques: le 2 arachidonyl glycerol (2AG) et l'anandamide (AEA) régulant l activité sécrétoire du monoxyde d azote (NO) par les cellules microgliales du SNC. A l inverse des vertébrés, Hirudo est capable de réguler très finement sa libération de NO à l endroit lésé du SNC ce qui favorise les mécanismes de réparation cellulaire après lésion. En parallèle, une nouvelle approche de développement de l'imagerie par spectrométrie de masse MALDI Tof-Tof des lipides a permis pour la première fois, de valider l'utilisation de colorants histologiques lipophiles en vue de localiser et de caractériser des lipides in situ sur coupes de cerveaux de rat. Cette nouvelle approche par les colorants qualifiée de DALDI Tof-Tof (Dye-Assisted Laser Desorption Ionisation Time-of-fligh) a trouvé des applications directes dans le cancer de l'ovaire où une expression différentielle de lipides a été démontrée entre les régions saine et cancéreuse. Ce travail a donc permis d'une part de mettre en évidence une forte, implication des lipides de type cannabinoïdes sur le recrutement des cellules microgliales ainsi qu'une régulation de la production du NO au site de la lésion, et d'autre part, l'apparition de lipides spécifiques au sein des neurones au cours des phases de régénération nerveuse du SNC d'Hirudo. De plus, l'utilisation de colorants histologiques comme matrice en vue de détecter des lipides directement sur coupes de tissus permet d'associer l'application de l'imagerie MALDI des lipides à la recherche de marqueurs potentiels de nature lipidique dans le cancer de l'ovaire. Des perspectives d'interaction avec la biologie clinique et médicale pourraient être envisagées afin de compléter les diagnostics des anatomo-pathologistes par une localisation et une caractérisation des lipides impliqués dans une pathologie donnéeLipids are metabolites which can be declined in many classes. Most of them are known to figure as structural molecules in the cell membrane but a significant key role is also played as cellular messenger when interacting with specific membrane receptors. The leech Hirudo medicinalis which is a model for the neurosciences is able to regenerate its central nervous system (CNS) after a lesion in opposition with the CNS of vertebrate. In this way, we showed by Tof-SIMS imaging changes in the lipidome of harmed leech brain directly on tissue. In particular lipids such as fatty acids and triglycerides have been characterized during injuries of the leech CNS. These lipids were expressed in specific areas of damaged neurons. We also characterized endocannabinoids in crude lipid extracts of harmed leech brain by MALDI-Tof Tof mass spectrometry. The identified endocannabinoids anandamide (AEA) and 2-arachidonyl glycerol (2AG) are lipids which act as cellular messenger. During the injury of the leech brain, an increase in the 2AG concentration in the brain were demonstrated while AEA was decreasing. As a consequence, a modulation in the chemotactic effect on microglial cells was observed in the harmed connective. 2AG and AEA have also been demonstrated to regulate the NO release in activated microglia under neurotoxic rate. A new approach in developpemental research in lipidomic using MALDI imaging mass spectrometry allowed us to validate for the first time the use of specific histochemical dies for lipids as a way to detect and characterize lipids directly on rat brain tissue. This new approach of studiying lipids using dies named DALDI Tof-Tof (Dye-Assisted Laser Desorption Ionisation Time-of-fligh)has been successfully applied to the ovarian cancer where different patterns in the lipid content were detected on tissue slices between benign and malignant areas. This work highlighted in one hand the roles played by cannabinoids on the microglia chemotaxis and the regulation of NO secretion at the lesion site and in the second hand, the expression of specific lipids on neurons close to the lesion site during the regeneration process of the damaged leech CNS. Moreover, by using histological dies specific the the lipid staining as matrices, we showed that MALDI imaging of lipids allowed to detect lipids which could be potentially biomarkers for ovarian cancer. A cross-talk with scientist and medical fields can emerged since using both the same way to detect lipids on tissue for diagnosis purposes.LILLE1-Bib. Electronique (590099901) / SudocSudocFranceF

Molecular histoproteomy by MALDI mass spectrometry imaging to uncover markers of the impact of Nosema on Apis mellifera

International audienc

Comparative Proteomics Studies of Insect Cuticle by Tandem Mass Spectrometry: Application of a Novel Proteomics Approach to the Pea Aphid Cuticular Proteins

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Unraveling the Bombus terrestris Hemolymph, an Indicator of the Immune Response to Microbial Infections, through Complementary Mass Spectrometry Approaches

International audiencePollinators, including Bombus terrestris, are crucial for maintaining biodiversity in ecosystems and for agriculture. Deciphering their immune response under stress conditions is a key issue for protecting these populations. To assess this metric, we analyzed the B. terrestris hemolymph as an indicator of their immune status. Hemolymph analysis was carried out using mass spectrometry, MALDI molecular mass fingerprinting was used for its effectiveness in assessing the immune status, and high-resolution mass spectrometry was used to measure the impact of experimental bacterial infections on the “hemoproteome”. By infecting with three different types of bacteria, we observed that B. terrestris reacts in a specific way to bacterial attacks. Indeed, bacteria impact survival and stimulate an immune response in infected individuals, visible through changes in the molecular composition of their hemolymph. The characterization and label-free quantification of proteins involved in specific signaling pathways in bumble bees by bottom-up proteomics revealed differences in protein expression between the non-experimentally infected and the infected bees. Our results highlight the alteration of pathways involved in immune and defense reactions, stress, and energetic metabolism. Lastly, we developed molecular signatures reflecting the health status of B. terrestris to pave the way for diagnosis/prognosis tools in response to environmental stress

Insect mouthpart transcriptome unveils extension of cuticular protein repertoire and complex organization

International audienceSummary Insects have developed intriguing cuticles with very specific structures and functions, including microstructures governing their interactions with transmitted microbes, such in aphid mouthparts harboring virus receptors within such microstructures. Here, we provide the first transcriptome analysis of an insect mouthpart cuticle (“retort organs” ROs, the stylets’ precursors). This analysis defined stylets as a complex composite material. The retort transcriptome also allowed us to propose an algorithmic definition of a new cuticular protein (CP) family with low complexity and biased amino-acid composition. Finally, we identified a differentially expressed gene encoding a pyrokinin (PK) neuropeptide precursor and characterized the mandibular glands. Injection of three predicted synthetic peptides PK1/2/3 into aphids prior to ecdysis caused a molt-specific phenotype with altered head formation. Our study provides the most complete description to date of the potential protein composition of aphid stylets, which should improve the understanding of the transmission of stylet-borne viruses

Analysis of the retort organs, producing mouthpart cuticle, in (the pea) aphid

International audienceLes pucerons transmettent la plupart des virus végétaux, et pour une bonne partie de ces derniers (les virus non-circulants), les stylets de l’insecte sont les pièces anatomiques effectives de cette vection. Depuis uen dizaine d’année, la découverte d’un micro-organe spécifique à cette fonction, l’acrostyle, intéresse particulièrement les chercheusr en mécanismes moléculaires de la vection. En effet, comme cela a été prouvé dasn les deux dernières années, cet acrostyle porte les déterminants moléculaires (récepteurs) de la transmission par puceron de plusieusr de ces phytovirus, le CaMV notamment.Dans le cadre du projet ANR StylHook, un module de travail (WP1) a porté sur la caractérisation exhaustive des proteins de l’acrostyle et des stylets du puceron du pois, dont le génome est identifié et correctement annoté depuis 2010. Ce travail analytique sur les protéines cuticulaires (structurales) des pucerons a été lancé par une approche quadruple:1: Caractérisation (immuno-)histologique et cellulaire de l’organe responsable de la synthèse périodique des stylets lors de la mue de l’insecte (la glande cuticulaire appelée « retort  organ », ou « organe alambiqué »…2: Caractérisation protéomique des stylets, et tentative de caractérisation de l’acrostyle par la même approche et microdissection laser3: Caréctrisation protéomique des glandes des stylets (retort organ, glandes maxillaires et mandibulaires)4: Caractérisation transcriptomique de l’organ retort au momùent du pic de transcription pre-ecdysial (pic de synthèse des stylets adultes).Ces résultats ont permis une caractérisation très exhaustive (couverture totale) de la composition cuticulaire protéique des stylets, une identification des quelques différences (protéomique, pas transcriptomique) entre stylets maxillaires et mandibulaires.Ils nous ont permis de montrer que ce matériau était pour l’heure le plus complexe des matériaux cuticulaires analysés jusqu’à présent (cuticule larvaire de lépidoptères, ailes de coléoptères ou de diptères), et nosu ont permis de proposer une méthode non-homologique pour la prédiction de nouvelles classes de protéines cuticulaires chez les insectes, basées sur le biais compositionnel et non sur les algorithmes d’identification/classification basés sur des régions conservées (HMM) classiqument utilisé jusque-là

MALDI–MS Profiling to Address Honey Bee Health Status under Bacterial Challenge through Computational Modeling

International audienceHoney bees play a critical role in the maintenance of plant biodiversity and sustainability of food webs. In the past few decades, bees have been subjected to biotic and abiotic threats causing various colony disorders. Therefore, monitoring solutions to help beekeepers to improve bee health are necessary. Matrix-assisted laser desorption ionization–mass spectrometry (MALDI–MS) profiling has emerged within this decade as a powerful tool to identify in routine micro-organisms and is currently used in real-time clinical diagnosis. MALDI BeeTyping is developed to monitor significant hemolymph molecular changes in honey bees upon infection with a series of entomopathogenic Gram-positive and -negative bacteria. A Serratia marcescens strain isolated from one naturally infected honey bee collected from the field is also considered. A series of hemolymph molecular mass fingerprints is individually recorded and to the authors’ knowledge, the first computational model harboring a predictive score of 97.92% and made of nine molecular signatures that discriminate and classify the honey bees’ systemic response to the bacteria is built. Hence, the model is challenged by classifying a training set of hemolymphs and an overall recognition of 91.93% is obtained. Through this work, a novel, time and cost saving high-throughput strategy that addresses honey bee health on an individual scale is introduced