A Toll pathway effector protects Drosophila specifically from distinct toxins secreted by a fungus or a bacterium.

The Drosophila systemic immune response against many Gram-positive bacteria and fungi is mediated by the Toll pathway. How Toll-regulated effectors actually fulfill this role remains poorly understood as the known Toll-regulated antimicrobial peptide (AMP) genes are active only against filamentous fungi and not against Gram-positive bacteria or yeasts. Besides AMPs, two families of peptides secreted in response to infectious stimuli that activate the Toll pathway have been identified, namely Bomanins and peptides derived from a polyprotein precursor known as Baramicin A (BaraA). Unexpectedly, the deletion of a cluster of 10 Bomanins phenocopies the Toll mutant phenotype of susceptibility to infections. Here, we demonstrate that BaraA is required specifically in the host defense against Enterococcus faecalis and against the entomopathogenic fungus Metarhizium robertsii, albeit the fungal burden is not altered in BaraA mutants. BaraA protects the fly from the action of distinct toxins secreted by these Gram-positive and fungal pathogens, respectively, Enterocin V and Destruxin A. The injection of Destruxin A leads to the rapid paralysis of flies, whether wild type (WT) or mutant. However, a larger fraction of wild-type than BaraA flies recovers from paralysis within 5 to 10 h. BaraAs' function in protecting the host from the deleterious action of Destruxin is required in glial cells, highlighting a resilience role for the Toll pathway in the nervous system against microbial virulence factors. Thus, in complement to the current paradigm, innate immunity can cope effectively with the effects of toxins secreted by pathogens through the secretion of dedicated peptides, independently of xenobiotics detoxification pathways.

The Toll pathway mediates Drosophila resilience to Aspergillus mycotoxins through specific Bomanins.

Host defense against infections encompasses both resistance, which targets microorganisms for neutralization or elimination, and resilience/disease tolerance, which allows the host to withstand/tolerate pathogens and repair damages. In Drosophila, the Toll signaling pathway is thought to mediate resistance against fungal infections by regulating the secretion of antimicrobial peptides, potentially including Bomanins. We find that Aspergillus fumigatus kills Drosophila Toll pathway mutants without invasion because its dissemination is blocked by melanization, suggesting a role for Toll in host defense distinct from resistance. We report that mutants affecting the Toll pathway or the 55C Bomanin locus are susceptible to the injection of two Aspergillus mycotoxins, restrictocin and verruculogen. The vulnerability of 55C deletion mutants to these mycotoxins is rescued by the overexpression of Bomanins specific to each challenge. Mechanistically, flies in which BomS6 is expressed in the nervous system exhibit an enhanced recovery from the tremors induced by injected verruculogen and display improved survival. Thus, innate immunity also protects the host against the action of microbial toxins through secreted peptides and thereby increases its resilience to infection.

Application of a robust MALDI mass spectrometry approach for bee pollen investigation.

Pollen collected by pollinators can be used as a marker of the foraging behavior as well as indicate the botanical species present in each environment. Pollen intake is essential for pollinators' health and survival. During the foraging activity, some pollinators, such as honeybees, manipulate the collected pollen mixing it with salivary secretions and nectar (corbicular pollen) changing the pollen chemical profile. Different tools have been developed for the identification of the botanical origin of pollen, based on microscopy, spectrometry, or molecular markers. However, up to date, corbicular pollen has never been investigated. In our work, corbicular pollen from 5 regions with different climate conditions was collected during spring. Pollens were identified with microscopy-based techniques, and then analyzed in MALDI-MS. Four different chemical extraction solutions and two physical disruption methods were tested to achieve a MALDI-MS effective protocol. The best performance was obtained using a sonication disruption method after extraction with acetic acid or trifluoroacetic acid. Therefore, we propose a new rapid and reliable methodology for the identification of the botanical origin of the corbicular pollens using MALDI-MS. This new approach opens to a wide range of environmental studies spanning from plant biodiversity to ecosystem trophic interactions.

Proteomics and Immune Response Differences in Apis mellifera and Apis cerana Inoculated with Three Nosema ceranae Isolates.

Nosema ceranae infects midgut epithelial cells of the Apis species and has jumped from its original host A. cerana to A. mellifera worldwide, raising questions about the response of the new host. We compared the responses of these two species to N. ceranae isolates from A. cerana, A. mellifera from Thailand and A. mellifera from France. Proteomics and transcriptomics results were combined to better understand the impact on the immunity of the two species. This is the first combination of omics analyses to evaluate the impact of N. ceranae spores from different origins and provides new insights into the differential immune responses in honeybees inoculated with N. ceranae from original A. cerana. No difference in the antimicrobial peptides (AMPs) was observed in A. mellifera, whereas these peptides were altered in A. cerana compared to controls. Inoculation of A. mellifera or A. cerana with N. ceranae upregulated AMP genes and cellular-mediated immune genes but did not significantly alter apoptosis-related gene expression. A. cerana showed a stronger immune response than A. mellifera after inoculation with different N. ceranae isolates. N. ceranae from A. cerana had a strong negative impact on the health of A. mellifera and A. cerana compared to other Nosema isolates.

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

Pollinators, 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.

Honey Bee Larval Hemolymph as a Source of Key Nutrients and Proteins Offers a Promising Medium for Varroa destructor Artificial Rearing.

Varroa destructor, a major ectoparasite of the Western honey bee Apis mellifera, is a widespread pest that damages colonies in the Northern Hemisphere. Throughout their lifecycle, V. destructor females feed on almost every developmental stage of their host, from the last larval instar to the adult. The parasite is thought to feed on hemolymph and fat body, although its exact diet and nutritional requirements are poorly known. Using artificial Parafilm™ dummies, we explored the nutrition of V. destructor females and assessed their survival when fed on hemolymph from bee larvae, pupae, or adults. We compared the results with mites fed on synthetic solutions or filtered larval hemolymph. The results showed that the parasites could survive for several days or weeks on different diets. Bee larval hemolymph yielded the highest survival rates, and filtered larval plasma was sufficient to maintain the mites for 14 days or more. This cell-free solution therefore theoretically contains all the necessary nutrients for mite survival. Because some bee proteins are known to be hijacked without being digested by the parasite, we decided to run a proteomic analysis of larval honey bee plasma to highlight the most common proteins in our samples. A list of 54 proteins was compiled, including several energy metabolism proteins such as Vitellogenin, Hexamerin, or Transferrins. These molecules represent key nutrient candidates that could be crucial for V. destructor survival.

Parasite and Pesticide Impacts on the Bumblebee (Bombus terrestris) Haemolymph Proteome.

Pesticides pose a potential threat to bee health, especially in combination with other stressors, such as parasites. However, pesticide risk assessment tests pesticides in isolation from other stresses, i.e., on otherwise healthy bees. Through molecular analysis, the specific impacts of a pesticide or its interaction with another stressor can be elucidated. Molecular mass profiling by MALDI BeeTyping® was used on bee haemolymph to explore the signature of pesticidal and parasitic stressor impacts. This approach was complemented by bottom-up proteomics to investigate the modulation of the haemoproteome. We tested acute oral doses of three pesticides-glyphosate, Amistar and sulfoxaflor-on the bumblebee Bombus terrestris, alongside the gut parasite Crithidia bombi. We found no impact of any pesticide on parasite intensity and no impact of sulfoxaflor or glyphosate on survival or weight change. Amistar caused weight loss and 19-41% mortality. Haemoproteome analysis showed various protein dysregulations. The major pathways dysregulated were those involved in insect defences and immune responses, with Amistar having the strongest impact on these dysregulated pathways. Our results show that even when no response can be seen at a whole organism level, MALDI BeeTyping® can detect effects. Mass spectrometry analysis of bee haemolymph provides a pertinent tool to evaluate stressor impacts on bee health, even at the level of individuals.

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

Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) is a powerful technology used to investigate the spatio-temporal distribution of a huge number of molecules throughout a body/tissue section. In this paper, we report the use of MALDI IMS to follow the molecular impact of an experimental infection of Apis mellifera with the microsporidia Nosema ceranae. We performed representative molecular mass fingerprints of selected tissues obtained by dissection. This was followed by MALDI IMS workflows optimization including specimen embedding and positioning as well as washing and matrix application. We recorded the local distribution of peptides/proteins within different tissues from experimentally infected versus non infected honeybees. As expected, a distinction in these molecular profiles between the two conditions was recorded from different anatomical sections of the gut tissue. More importantly, we observed differences in the molecular profiles in the brain, thoracic ganglia, hypopharyngeal glands, and hemolymph. We introduced MALDI IMS as an effective approach to monitor the impact of N. ceranae infection on A. mellifera. This opens perspectives for the discovery of molecular changes in peptides/proteins markers that could contribute to a better understanding of the impact of stressors and toxicity on different tissues of a bee in a single experiment.

Deciphering the molecular mechanisms of mother-to-egg immune protection in the mealworm beetle Tenebrio molitor.

In a number of species, individuals exposed to pathogens can mount an immune response and transmit this immunological experience to their offspring, thereby protecting them against persistent threats. Such vertical transfer of immunity, named trans-generational immune priming (TGIP), has been described in both vertebrates and invertebrates. Although increasingly studied during the last decade, the mechanisms underlying TGIP in invertebrates are still elusive, especially those protecting the earliest offspring life stage, i.e. the embryo developing in the egg. In the present study, we combined different proteomic and transcriptomic approaches to determine whether mothers transfer a "signal" (such as fragments of infecting bacteria), mRNA and/or protein/peptide effectors to protect their eggs against two natural bacterial pathogens, namely the Gram-positive Bacillus thuringiensis and the Gram-negative Serratia entomophila. By taking the mealworm beetle Tenebrio molitor as a biological model, our results suggest that eggs are mainly protected by an active direct transfer of a restricted number of immune proteins and of antimicrobial peptides. In contrast, the present data do not support the involvement of mRNA transfer while the transmission of a "signal", if it happens, is marginal and only occurs within 24h after maternal exposure to bacteria. This work exemplifies how combining global approaches helps to disentangle the different scenarios of a complex trait, providing a comprehensive characterization of TGIP mechanisms in T. molitor. It also paves the way for future alike studies focusing on TGIP in a wide range of invertebrates and vertebrates to identify additional candidates that could be specific to TGIP and to investigate whether the TGIP mechanisms found herein are specific or common to all insect species.

Functional Diversification of Oyster Big Defensins Generates Antimicrobial Specificity and Synergy against Members of the Microbiota.

Big defensins are two-domain antimicrobial peptides (AMPs) that have highly diversified in mollusks. Cg-BigDefs are expressed by immune cells in the oyster Crassostrea gigas, and their expression is dampened during the Pacific Oyster Mortality Syndrome (POMS), which evolves toward fatal bacteremia. We evaluated whether Cg-BigDefs contribute to the control of oyster-associated microbial communities. Two Cg-BigDefs that are representative of molecular diversity within the peptide family, namely Cg-BigDef1 and Cg-BigDef5, were characterized by gene cloning and synthesized by solid-phase peptide synthesis and native chemical ligation. Synthetic peptides were tested for antibacterial activity against a collection of culturable bacteria belonging to the oyster microbiota, characterized by 16S sequencing and MALDI Biotyping. We first tested the potential of Cg-BigDefs to control the oyster microbiota by injecting synthetic Cg-BigDef1 into oyster tissues and analyzing microbiota dynamics over 24 h by 16S metabarcoding. Cg-BigDef1 induced a significant shift in oyster microbiota ß-diversity after 6 h and 24 h, prompting us to investigate antimicrobial activities in vitro against members of the oyster microbiota. Both Cg-BigDef1 and Cg-BigDef5 were active at a high salt concentration (400 mM NaCl) and showed broad spectra of activity against bacteria associated with C. gigas pathologies. Antimicrobial specificity was observed for both molecules at an intra- and inter-genera level. Remarkably, antimicrobial spectra of Cg-BigDef1 and Cg-BigDef5 were complementary, and peptides acted synergistically. Overall, we found that primary sequence diversification of Cg-BigDefs has generated specificity and synergy and extended the spectrum of activity of this peptide family.

Proteomics of Anatomical Sections of the Gut of Nosema-Infected Western Honeybee (Apis mellifera) Reveals Different Early Responses to Nosema spp. Isolates.

Honeybees play an important role in pollinating native plants and agricultural crops and produce valuable hive products. Within the last decade, honeybee colonies have been reported to be in decline, due to both biotic and abiotic stress factors including pathogens and pesticides. This study evaluated the impact of different isolates of Nosema spp. [Nosema apis spores (NA), Nosema ceranae from Apis mellifera from France (NF), N. ceranae from Apis cerana from Thailand (NC1), and N. ceranae from A. mellifera from Thailand (NC2)] on the different gut sections of newly emerged adult A. mellifera bees. With an attempt to decipher the early impact of Nosema spp. on the first barrier against Nosema infection, we used off-gel bottom-up proteomics on the different anatomical sections of the gut four days post inoculation. A total of 2185 identified proteins in the esophagus, 2095 in the crop, 1571 in the midgut, 2552 in the ileum, and 3173 in the rectum were obtained. Using label-free quantification, we observed that the response of the host varies according to the Nosema spp. (N. apis versus N. ceranae) and the geographical origin of Nosema. The proteins in the midgut of A. mellifera, orally inoculated with spores of N. ceranae isolated from France, were the most altered, when compared with controls, exhibiting 50 proteins down-regulated and 16 up-regulated. We thereby established the first mass-spectrometry-based proteomics of different anatomical sections of the gut tissue of Nosema-infected A. mellifera four days post inoculation, following infection by different isolates of Nosema spp. that provoked differential host responses. We reported an alteration of proteins involved in the metabolic pathways and specifically eight proteins of the oxidative phosphorylation pathway. More importantly, we propose that the collagen IV NC1 domain-containing protein may represent an early prognostic marker of the impact of Nosema spores on the A. mellifera health status. Data are available via ProteomeXchange with the identifier PXD021848.

Transcriptomic, proteomic and ultrastructural studies on salinity-tolerant Aedes aegypti in the context of rising sea levels and arboviral disease epidemiology.

BACKGROUND: Aedes aegypti mosquito, the principal global vector of arboviral diseases, lays eggs and undergoes larval and pupal development to become adult mosquitoes in fresh water (FW). It has recently been observed to develop in coastal brackish water (BW) habitats of up to 50% sea water, and such salinity tolerance shown to be an inheritable trait. Genomics of salinity tolerance in Ae. aegypti has not been previously studied, but it is of fundamental biological interest and important for controlling arboviral diseases in the context of rising sea levels increasing coastal ground water salinity. RESULTS: BW- and FW-Ae. aegypti were compared by RNA-seq analysis on the gut, anal papillae and rest of the carcass in fourth instar larvae (L4), proteomics of cuticles shed when L4 metamorphose into pupae, and transmission electron microscopy of cuticles in L4 and adults. Genes for specific cuticle proteins, signalling proteins, moulting hormone-related proteins, membrane transporters, enzymes involved in cuticle metabolism, and cytochrome P450 showed different mRNA levels in BW and FW L4 tissues. The salinity-tolerant Ae. aegypti were also characterized by altered L4 cuticle proteomics and changes in cuticle ultrastructure of L4 and adults. CONCLUSIONS: The findings provide new information on molecular and ultrastructural changes associated with salinity adaptation in FW mosquitoes. Changes in cuticles of larvae and adults of salinity-tolerant Ae. aegypti are expected to reduce the efficacy of insecticides used for controlling arboviral diseases. Expansion of coastal BW habitats and their neglect for control measures facilitates the spread of salinity-tolerant Ae. aegypti and genes for salinity tolerance. The transmission of arboviral diseases can therefore be amplified in multiple ways by salinity-tolerant Ae. aegypti and requires appropriate mitigating measures. The findings in Ae. aegypti have attendant implications for the development of salinity tolerance in other fresh water mosquito vectors and the diseases they transmit.

Defect in the nuclear pore membrane glycoprotein 210-like gene is associated with extreme uncondensed sperm nuclear chromatin and male infertility: a case report.

After the two meiotic divisions, haploid round spermatids undergo dramatic changes to become mature spermatozoa. One of the main transformations consists of compacting the cell nucleus to confer the sperm its remarkable hydrodynamic property and to protect its DNA from the oxidative stress it will encounter during its reproductive journey. Here, we studied an infertile subject with low sperm count, poor motility and highly abnormal spermatozoa with strikingly large heads due to highly uncondensed nuclear sperm DNA. Whole-exome sequencing was performed on the subject's DNA to identify the genetic defect responsible for this severe sperm anomaly. Bioinformatics analysis of exome sequence data uncovered a homozygous loss of function variant, ENST00000368559.7:c.718-1G>A, altering a consensus splice site expected to prevent the synthesis of the nucleoporin 210 like (NUP210L) protein. High-resolution mass spectrometry of sperm protein extracts did not reveal any NUP210L peptide sequence in the patient's sperm, contrary to what was observed in control donors, thus confirming the absence of NUP210L in the patient's sperm. Interestingly, homozygous Nup210l knock-out mice have been shown to be infertile due to a reduced sperm count, a high proportion of round-headed sperm, other head and flagella defects and a poor motility. NUP210L is almost exclusively expressed in the testis and sequence analogy suggests that it encodes a nuclear pore membrane glycoprotein. The protein might be crucial to regulate nuclear trafficking during and/or before spermiogenesis, its absence potentially impeding adequate nuclear compaction by preventing the entry of histone variants/transition proteins/protamines into the nucleus and/or by preventing the adequate replacement of core histones. This work describes a new gene necessary for male fertility, potentially improving the efficiency of the genetic diagnosis of male infertility. The function of NUP210L still remains to be resolved and its future investigation will help to understand the complex mechanisms necessary for sperm compaction.

Matrix-assisted laser desorption/ionization mass spectrometry biotyping, an approach for deciphering and assessing the identity of the honeybee pathogen Nosema.

RATIONALE: The microsporidia are obligate intracellular pathogenic fungi that parasitize a wide range of invertebrate and vertebrate hosts and have important impacts on health, food security and the economy. In this paper, we focus on Nosema ceranae and N. apis, which chronically infect the digestive tract of honeybees, altering their physiology and lifespan. METHODS: We applied matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) for rapid molecular profiling of extracts of Nosema spores in order to identify the species and the geographical origin, and assess the viability status of Nosema microsporidia in conjunction with a flow cytometric approach. Pure solutions of spores were prepared for flow cytometric analysis and MALDI-MS profiling. A mechanical extraction of viable or heat-killed Nosema spores was conducted to obtain mass fingerprints of peptides/proteins for samples of microsporidia from different geographical origins (MBO.NC01, MBO.NC02 and MBO.NA01). RESULTS: A distinction in the peptide/protein profiles between two isolates with different geographical origins was observed. Mass fingerprints of viable and experimentally killed spores were also clearly distinguishable, regardless of Nosema species. Finally, using our computational models on the different Nosema species, we were able to classify five independent isolates of Nosema microsporidia. CONCLUSIONS: We have shown that MALDI-MS is a rapid, cost-effective and simple method for identifying Nosema species. We demonstrated that MALDI Biotyping could represent a valuable surveillance tool of nosemosis in apiaries for sanitary services and beekeepers.

Impact of an Antifungal Insect Defensin on the Proteome of the Phytopathogenic Fungus Botrytis cinerea.

ETD151, an analogue of the antifungal insect defensin heliomicin, is an antifungal peptide active against yeasts and filamentous fungi. To decipher the mechanisms underlying its molecular action on the phytopathogenic fungus Botrytis cinerea, a necrotrophic pathogen responsible for gray mold disease, we investigated the changes in 3 day-old mycelia upon treatment with different concentrations of ETD151. Optical and fluorescence microscopies were used prior to establishing the peptide/protein profiles through two mass spectrometry approaches: MALDI profiling, to generate molecular mass fingerprints as peptide signatures, and a gel-free bottom-up proteomics approach. Our results show that a concentration of ETD151 above the half-maximal inhibitory concentration can alter the integrity of the mycelial structure of B. cinerea. Furthermore, reproducible modifications of the peptide/protein composition were demonstrated in the presence of ETD151 within a 1500-16,000 mass (m/z) range. After the robustness of LC-ESI-MS/MS analysis on B. cinerea mycelial extracts was confirmed, our analyses highlighted 340 significantly modulated proteins upon treatment with ETD151 within a 4.8-466 kDa mass range. Finally, data mapping on KEGG pathways revealed the molecular impact of ETD151 on at least six pathways, namely, spliceosome, ribosome, protein processing in endoplasmic reticulum, endocytosis, MAPK signaling pathway, and oxidative phosphorylation.

Cuticular Structure Proteomics in the Pea Aphid Acyrthosiphon pisum Reveals New Plant Virus Receptor Candidates at the Tip of Maxillary Stylets.

Aphids are phloem-feeding insects known as major pests in agriculture that are able to transmit hundreds of plant viruses. The majority of these viruses, classified as noncirculative, are retained and transported on the inner surface of the cuticle of the needle-like mouthparts while the aphids move from plant to plant. Identification of receptors of viruses within insect vectors is a key challenge because they are promising targets for alternative control strategies. The acrostyle, an organ discovered earlier within the common food/salivary canal at the tip of aphid maxillary stylets, displays proteins at the cuticle-fluid interface, some of which are receptors of noncirculative viruses. To assess the presence of stylet- and acrostyle-specific proteins and identify putative receptors, we have developed a comprehensive comparative analysis of the proteomes of four cuticular anatomical structures of the pea aphid, stylets, antennae, legs, and wings. In addition, we performed systematic immunolabeling detection of the cuticular proteins identified by mass spectrometry in dissected stylets. We thereby establish the first proteome of stylets of an insect and determine the minimal repertoire of the cuticular proteins composing the acrostyle. Most importantly, we propose a short list of plant virus receptor candidates, among which RR-1 proteins are remarkably predominant. The data are available via ProteomeXchange (PXD016517).

Identification, Characterization and Synthesis of Walterospermin, a Sperm Motility Activator from the Egyptian Black Snake Walterinnesia aegyptia Venom.

Animal venoms are small natural mixtures highly enriched in bioactive components. They are known to target at least two important pharmacological classes of cell surface receptors: ion channels and G protein coupled receptors. Since sperm cells express a wide variety of ion channels and membrane receptors, required for the control of cell motility and acrosome reaction, two functions that are defective in infertility issues, animal venoms should contain interesting compounds capable of modulating these two essential physiological functions. Herein, we screened for bioactive compounds from the venom of the Egyptian black snake Walterinnesia aegyptia (Wa) that possess the property to activate sperm motility in vitro from male mice OF1. Using RP-HPLC and cation exchange chromatography, we identified a new toxin of 6389.89 Da (termed walterospermin) that activates sperm motility. Walterospermin was de novo sequenced using a combination of matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF/TOF MS/MS) and liquid chromatography electrospray ionization quadrupole time-of-flight mass spectrometry (LC-ESI-QTOF MS/MS) following reduction, alkylation, and enzymatic proteolytic digestion with trypsin, chymotrypsin or V8 protease. The peptide is 57 amino acid residues long and contains three disulfide bridges and was found to be identical to the previously cloned Wa Kunitz-type protease inhibitor II (Wa Kln-II) sequence. Moreover, it has strong homology with several other hitherto cloned Elapidae and Viperidae snake toxins suggesting that it belongs to a family of compounds able to regulate sperm function. The synthetic peptide shows promising activation of sperm motility from a variety of species, including humans. Its fluorescently-labelled analog predominantly marks the flagellum, a localization in agreement with a receptor that controls motility function.

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

Honey 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.

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

The cuticle is a biological composite material consisting principally of N-acetylglucosamine polymer embedded in cuticular proteins (CPs). CPs have been studied and characterized by mass spectrometry in several cuticular structures and in many arthropods. Such analyses were carried out by protein extraction using SDS followed by electrophoresis, allowing detection and identification of numerous CPs. To build a repertoire of cuticular structures from Bombyx mori, Apis mellifera and Anopheles gambiae the use of SDS and electrophoresis was avoided. Using the combination of hexafluoroisopropanol and of a surfactant compatible with MS, a high number of CPs was identified in An. gambiae wings, legs and antennae, and in the thoracic integument cuticle of Ap. mellifera pupae. The exoskeleton analysis of B. mori larvae allowed to identify 85 CPs from a single larva. Finally, the novel proteomics approach was tested on cuticles left behind after the molt from the fourth instar of Acyrthosiphon pisum. Analysis of these cast cuticles allowed to identify 100 Ac. pisum CPs as authentic cuticle constituents. These correspond to 68% of the total putative CPs previously annotated for this pea aphid. While this paper analyzes only the recovered cuticular proteins, peptides from many other proteins were also detected.

Spermaurin, an La1-like peptide from the venom of the scorpion Scorpio maurus palmatus, improves sperm motility and fertilization in different mammalian species.

STUDY QUESTION: Is it possible to identify original compounds that are able to enhance sperm motility from the venom of the scorpion Scorpio maurus palmatus? SUMMARY ANSWER: We identified a potent disulfide-rich peptide (DRP) of 73 amino acids that significantly improved the motility of fresh and frozen-thawed sperm in different mammalian species, including human, and improved fertilization outcome in mouse IVF experiments. WHAT IS KNOWN ALREADY: Any disturbance of sperm motility has a strong impact on fertilization and can lead to subfertility or infertility. Significant efforts have, therefore,  been made to identify pharmacological drugs that might improve sperm motility. Such compounds are particularly useful in azoospermia to improve testicular sperm extraction and in the domain of cryopreservation because the motility of frozen-thawed sperm is reduced. STUDY DESIGN, SIZE, DURATION: This was a basic science/medical research study aimed at identifying original compounds from a library of venoms able to enhance mammalian sperm motility, including human. We first identified in the venom of a scorpion S. m. palmatus a fraction able to potently activate sperm motility. We next purified and characterized the compound by liquid chromatography, mass spectrometry and peptide synthesis. Finally, the potency and toxicity of both purified and synthetic versions of the identified compound on sperm motility were assessed using different in vitro tests in different mammalian species. PARTICIPANTS/MATERIALS, SETTING, METHODS: For human sperm, biological samples were collected from normozoospermic donors and subfertile patients attending a reproduction department for diagnostic semen analysis. Testicular sperm was collected from cynomolgus monkeys (Macaca fascicularis) euthanized for the needs of specific authorized research projects. The peptide was also tested on bovine and mouse epidydimal sperm. We measured different sperm motility parameters with a computer-assisted sperm analysis system in the presence or absence of the peptide. MAIN RESULTS AND THE ROLE OF CHANCE: Size exclusion chromatography enabled us to isolate a fraction of the venom of S. m. palmatus able to increase sperm motility. By liquid chromatography and mass spectrometry, a peptide comprising 73 amino acids with 4 disulfide bridges was identified as responsible for the biological activity and called 'spermaurin'. The identity of spermaurin was confirmed by chemical synthesis. We showed that the peptide increased the motility of fresh and frozen-thawed human sperm. We observed that the potency of the peptide was higher on fresh ejaculated spermatozoa with a low motility, achieving a 100% increase of curvilinear velocity in poorly performing sperm. We also demonstrated that peptide is effective on bovine and mouse fresh epididymal, bovine frozen-thawed ejaculated and fresh non-human primate testicular sperm. Finally, in mouse IVF, the production of 2-cell embryos was increased by 24% when sperm were treated with the peptide. LIMITATIONS, REASONS FOR CAUTION: This work is an in vitro evaluation of the ability of spermaurin to improve sperm motility parameters. Another limitation of this study is the small number of human sperm samples tested with the natural (n = 36) and synthetic (n = 12) peptides. Moreover, the effect of the peptide on IVF outcome was only tested in mouse and further tests with human and bovine gametes are required to confirm and extend this result in other mammalian species. WIDER IMPLICATIONS OF THE FINDINGS: This work confirms our initial study showing that venoms represent an interesting source of molecules that are able to modify sperm physiology. Moreover, this work presents the first demonstrated biological action of a venom peptide from the scorpion S. m. palmatus with sequence similarities to La1 peptide from Liocheles australasiae (Wood scorpion), a widespread family of DRPs. LARGE SCALE DATA: Not applicable. STUDY FUNDING/COMPETING INTEREST(S): This work is part of the project 'LAB COM-14 LAB7 0004 01-LIPAV', funded by the program LabCom 2014 from the French Research Agency (ANR). Dr Arnoult reports grants from IMV Technologies during the conduct of the study. In addition, Drs Arnoult, Martinez, Ray and Schmitt have a patent EP16305642.7 pending containing some of the information presented in this manuscript.