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.

A blood test to monitor bee health across a European network of agricultural sites of different land-use by MALDI BeeTyping mass spectrometry.

There are substantial concerns about impaired honey bee health and colony losses due to several poorly understood factors. We used MALDI profiling (MALDI BeeTyping®) analysis to investigate how some environmental and management factors under field conditions across Europe affected the honey bee haemolymph peptidome (all peptides in the circulatory fluid), as a profile of molecular markers representing the immune status of Apis mellifera. Honey bees were exposed to a range of environmental stressors in 128 agricultural sites across eight European countries in four biogeographic zones, with each country contributing eight sites each for two different cropping systems: oilseed rape (OSR) and apple (APP). The full haemolymph peptide profiles, including the presence and levels of three key immunity markers, namely the antimicrobial peptides (AMPs) Apidaecin, Abaecin and Defensin-1, allowed the honey bee responses to environmental variables to be discriminated by country, crop type and site. When considering just the AMPs, it was not possible to distinguish between countries by the prevalence of each AMP in the samples. However, it was possible to discriminate between countries on the amounts of the AMPs, with the Swedish samples in particular expressing high amounts of all AMPs. A machine learning model was developed to discriminate the haemolymphs of bees from APP and OSR sites. The model was 90.6 % accurate in identifying the crop type from the samples used to build the model. Overall, MALDI BeeTyping® of bee haemolymph represents a promising and cost-effective "blood test" for simultaneously monitoring dozens of peptide markers affected by environmental stressors at the landscape scale, thus providing policymakers with new diagnostic and regulatory tools for monitoring bee health.

The Response of the Honey Bee Gut Microbiota to Nosema ceranae Is Modulated by the Probiotic Pediococcus acidilactici and the Neonicotinoid Thiamethoxam.

The honey bee Apis mellifera is exposed to a variety of biotic and abiotic stressors, such as the highly prevalent microsporidian parasite Nosema (Vairimorpha) ceranae and neonicotinoid insecticides. Both can affect honey bee physiology and microbial gut communities, eventually reducing its lifespan. They can also have a combined effect on the insect's survival. The use of bacterial probiotics has been proposed to improve honey bee health, but their beneficial effect remains an open question. In the present study, western honey bees were experimentally infected with N. ceranae spores, chronically exposed to the neonicotinoid thiamethoxam, and/or supplied daily with the homofermentative bacterium Pediococcus acidilactici MA18/5M thought to improve the honey bees' tolerance to the parasite. Deep shotgun metagenomic sequencing allowed the response of the gut microbiota to be investigated with a taxonomic resolution at the species level. All treatments induced significant changes in honey bee gut bacterial communities. Nosema ceranae infection increased the abundance of Proteus mirabilis, Frischella perrara, and Gilliamella apicola and reduced the abundance of Bifidobacterium asteroides, Fructobacillus fructosus, and Lactobacillus spp. Supplementation with P. acidilactici overturned some of these alterations, bringing back the abundance of some altered species close to the relative abundance found in the controls. Surprisingly, the exposure to thiamethoxam also restored the relative abundance of some species modulated by N. ceranae. This study shows that stressors and probiotics may have an antagonistic impact on honey bee gut bacterial communities and that P. acidilactici may have a protective effect against the dysbiosis induced by an infection with N. ceranae.

Exploring the potential of Brazilian Amazonian scorpion venoms: A comprehensive review of research from 2001 to 2021.

The Amazon biome is home to many scorpion species, with around two hundred identified in the region. Of these, forty-eight species have been reported in Brazil so far and six of them are of medical importance: Tityus apiacas, T. metuendus, T. obscurus, T. raquelae, T. silvestris, and T. strandi. Three non-medically important species have also been studied: Opisthanthuscayaporum, Brotheas amazonicus and Rhopalurus laticauda. The venom of the scorpion T. obscurus is the most studied, followed by O. cayaporum. We aim to update the study of these Amazonian scorpion species. We will explore the harmful and beneficial properties of scorpion venom toxins and how they could be applied in drug development. This systematic review will focus on collecting and analyzing venoms from scorpions in Brazil. Only papers on Amazonian scorpion venom studies published between 2001 and 2021 (scientific articles, theses, and dissertations) were selected, based on the lists of scorpions available in the literature. Species found in the Amazon but not confirmed to be Brazilian were omitted from the review. Theses and dissertations were chosen over their derived articles. We found 42 eligible studies (13 theses, 27 articles and 2 patents) out of 17,950 studies and a basic statistical analysis was performed. The literature showed that T. obscurus was the most studied venom with 28 publications, followed by O. cayaporum with seven articles, B. amazonicus with four articles, T. metuendus with two article and R. laticauda with one article. No publication on the characterization of T. silvestris and T. apiacas venoms were found during the reviewed period, only the clinical aspects were covered. There is still much to be explored despite the increasing number of studies conducted in recent years. Amazonian scorpions have promising potential for pharmaceutical and clinical applications.

Molecular diversity assessed by MALDI mass spectrometry of two scorpion species venom from two different locations in Morocco.

Scorpion venom is a cocktail of molecules whose composition is remarkably plastic, controlled by several factors. The Moroccan scorpion fauna is characterized by its richness and high rate of endemism and the venom molecular variability of many species is not yet well characterized. The aim of the present study was to highlight the molecular variability of the venom composition of Androctonus amoreuxi and Buthacus stockmanni (endemic species), both belonging to the Buthidae family, collected from two Moroccan regions, Zagora and Tan-tan. Characterization of the molecular mass fingerprints (MFPs) of each specimen was performed by Matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) using a sandwich (Sand) and a dried-droplet (DD) sample preparation and dilutions. Considering these two methods, a total of 828 ion signals were detected, and Sand method produced more adducts (56%) than DD (44%). We observed interspecific variations in the venom composition between these two species showing they share 235 ion signals, while 226 and 367 are specific for these two species, respectively. Moreover, B. stockmanni specimens showed a clear difference in their MFPs between the two geographical areas studied, suggesting intraspecific variations. Moreover, specimens from each population also show an intraspecific variability. In addition, for the same individual, a variation in the venom composition was also recorded depending on the milking frequency. Our results confirmed the presence of characteristic components in each extracted venom sample. In conclusion, MFPs assessed by MALDI-MS represent a fast, non-supervised, sensitive, reliable and cost-efficient approach for taxonomic identification and molecular variability characterization. This study undoubtedly represents a step forward for understanding the scorpion venom plasticity, intra/inter variations, and their temporal and geographical variability.

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.

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.

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.

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.

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.

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.

Optimized Chemical Extraction Methods of Antimicrobial Peptides from Roots and Leaves of Extremophilic Plants: Anthyllis sericea and Astragalus armatus Collected from the Tunisian Desert.

Extraction methods depend mainly on the chemical nature of the extracted molecule. For these reasons, the selection of the extraction medium is a vital part of obtaining these molecules. The extraction of antimicrobial peptides (AMPs) from extremophile plants is important because of its potential pharmaceutical applications. This work focused on the evaluation of several solvents for the extraction of AMPs from the following two extremophile plants: Astragalus armatus and Anthyllis sericea from southern Tunisia. In order to identify the most efficient solvents and extraction solutions, we used sulfuric acid, dichloromethane, phosphate buffer, acetic acid and sodium acetate, and we tested them on leaves and roots of both the studied plants. The extracts obtained using sulfuric acid, dichloromethane and phosphate buffer extraction did not show any antimicrobial activity, whereas the acetic acid and sodium acetate extracts led to growth inhibition of some of the tested bacterial strains. The extracts of leaves and roots of An. sericea and As. armatus obtained by acetic acid and sodium acetate were proven to be active against Gram-positive bacteria and Gram-negative bacteria. Therefore, the most appropriate solvents to use for antimicrobial peptide extraction from both plants are acetic acid and sodium acetate.

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.

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.

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.

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.

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.

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.