Pollinator-assisted plant phenotyping, selection, and breeding for crop resilience to abiotic stresses.

Food security is threatened by climate change, with heat and drought being the main stresses affecting crop physiology and ecosystem services, such as plant-pollinator interactions. We hypothesize that tracking and ranking pollinators' preferences for flowers under environmental pressure could be used as a marker of plant quality for agricultural breeding to increase crop stress tolerance. Despite increasing relevance of flowers as the most stress sensitive organs, phenotyping platforms aim at identifying traits of resilience by assessing the plant physiological status through remote sensing-assisted vegetative indexes, but find strong bottlenecks in quantifying flower traits and in accurate genotype-to-phenotype prediction. However, as the transport of photoassimilates from leaves (sources) to flowers (sinks) is reduced in low-resilient plants, flowers are better indicators than leaves of plant well-being. Indeed, the chemical composition and amount of pollen and nectar that flowers produce, which ultimately serve as food resources for pollinators, change in response to environmental cues. Therefore, pollinators' preferences could be used as a measure of functional source-to-sink relationships for breeding decisions. To achieve this challenging goal, we propose to develop a pollinator-assisted phenotyping and selection platform for automated quantification of Genotype × Environment × Pollinator interactions through an insect geo-positioning system. Pollinator-assisted selection can be validated by metabolic, transcriptomic, and ionomic traits, and mapping of candidate genes, linking floral and leaf traits, pollinator preferences, plant resilience, and crop productivity. This radical new approach can change the current paradigm of plant phenotyping and find new paths for crop redomestication and breeding assisted by ecological decisions.

The heat is on: reduced detection of floral scents after heatwaves in bumblebees.

Global climate change disrupts key ecological processes and biotic interactions. The recent increase in heatwave frequency and severity prompts the evaluation of physiological processes that ensure the maintenance of vital ecosystem services such as pollination. We used experimental heatwaves to determine how high temperatures affect the bumblebees' ability to detect floral scents. Heatwaves induced strong reductions in antennal responses to floral scents in both tested bumblebee species (Bombus terrestris and Bombus pascuorum). These reductions were generally stronger in workers than in males. Bumblebees showed no consistent pattern of recovery 24 h after heat events. Our results suggest that the projected increased frequency and severity of heatwaves may jeopardize bumblebee-mediated pollination services by disrupting the chemical communication between plants and pollinators. The reduced chemosensitivity can decrease the bumblebees' abilities to locate food sources and lead to declines in colonies and populations.

Infection by Crithidia bombi increases relative abundance of Lactobacillus spp. in the gut of Bombus terrestris.

Gut microbial communities confer protection against natural pathogens in important pollinators from the genera Bombus and Apis. In commercial species B. terrestris and B. impatiens, the microbiota increases their resistance to the common and virulent trypanosomatid parasite Crithidia bombi. However, the mechanisms by which gut microorganisms protect the host are still unknown. Here, we test two hypotheses: microbiota protect the host (1) through stimulation of its immune response or protection of the gut epithelium and (2) by competing for resources with the parasite inside the gut. To test them, we reduced the microbiota of workers and then rescued the microbial community by feeding them with microbiota supplements. We then exposed them to an infectious dose of C. bombi and characterised gene expression and gut microbiota composition. We examined the expression of three antimicrobial peptide genes and Mucin-5AC, a gene with a putative role in gut epithelium protection, using qPCR. Although a protective effect against C. bombi was observed in bumblebees with supplemented microbiota, we did not observe an effect of the microbiota on gene expression that could explain alone the protective effect observed. On the other hand, we found an increased relative abundance of Lactobacillus bacteria within the gut of infected workers and a negative correlation of this genus with Gilliamella and Snodgrassella genera. Therefore, our results point to a displacement of bumblebee endosymbionts by C. bombi that might be caused by competition for space and nutrients between the parasite and the microbiota within the gut.

La microbiota intestinal confiere protección frente a los patógenos naturales en polinizadores importantes de los géneros Bombus y Apis. En concreto, la microbiota de las especies comerciales B. terrestris y B. impatients, incrementa su resistencia frente al parásito tripanosomátido común y virulento Crithidia bombi. Sin embargo, los mecanismos por los cuales los microorganismos protegen al hospedador todavía se desconocen. Aquí probamos dos hipótesis: la microbiota protege al hospedador (1) a través de la estimulación de la respuesta inmunitaria o la protección del epitelio y (2) por competición por los recursos con el parásito dentro del intestino. Para probar estas hipótesis, redujimos la microbiota de obreras y dimos suplementos de microbiota a una parte de ellas. Las expusimos a una dosis infecciosa de C. bombi y caracterizamos la expresión génica y la composición de la microbiota intestinal. Examinamos la expresión de los genes de tres péptidos antimicrobianos (AMPs) y de Mucin­5AC, un gen con un rol putativo en la protección del epitelio intestinal, usando la qPCR. Aunque observamos un efecto protector contra C. bombi en los abejorros suplementados con microbiota, no vimos un efecto en la expresión génica que pudiese explicar por sí solo la protección observada. Por otro lado, encontramos un incremento en la abundancia relativa de bacterias del género Lactobacillus en el intestino de obreras infectadas y una correlación negativa de este género con los géneros Gilliamella y Snodgrassella. Por tanto, nuestros resultados apuntan a un desplazamiento de los endosimbiontes por parte de C. bombi, que podría estar causado por la competición por espacio y nutrientes entre el parásito y la microbiota dentro del intestino.

Selection despite low genetic diversity and high gene flow in a rapid island invasion of the bumblebee, Bombus terrestris.

Invasive species are predicted to adjust their morphological, physiological and life-history traits to adapt to their non-native environments. Although a loss of genetic variation during invasion may restrict local adaptation, introduced species often thrive in novel environments. Despite being founded by just a few individuals, Bombus terrestris (Hymenoptera: Apidae) has in less than 30 years successfully spread across the island of Tasmania (Australia), becoming abundant and competitive with native pollinators. We use RADseq to investigate what neutral and adaptive genetic processes associated with environmental and morphological variation allow B. terrestris to thrive as an invasive species in Tasmania. Given the widespread abundance of B. terrestris, we expected little genetic structure across Tasmania and weak signatures of environmental and morphological selection. We found high gene flow with low genetic diversity, although with significant isolation-by-distance and spatial variation in effective migration rates. Restricted migration was evident across the mid-central region of Tasmania, corresponding to higher elevations, pastural land, low wind speeds and low precipitation seasonality. Tajima's D indicated a recent population expansion extending from the south to the north of the island. Selection signatures were found for loci in relation to precipitation, wind speed and wing loading. Candidate loci were annotated to genes with functions related to cuticle water retention and insect flight muscle stability. Understanding how a genetically impoverished invasive bumblebee has rapidly adapted to a novel island environment provides further understanding about the evolutionary processes that determine successful insect invasions, and the potential for invasive hymenopteran pollinators to spread globally.

Transcriptome-wide analysis uncovers regulatory elements of the antennal transcriptome repertoire of bumblebee at different life stages.

Bumblebees are crucial pollinators, providing essential ecosystem services and global food production. The success of pollination services relies on the interaction between sensory organs and the environment. The antenna functions as a versatile multi-sensory organ, pivotal in mediating chemosensory/olfactory information, and governs adaptive responses to environmental changes. Despite an increasing number of RNA-sequencing studies on insect antenna, comprehensive antennal transcriptome studies at the different life stages were not elucidated systematically. Here, we quantified the expression profile and dynamics of coding/microRNA genes of larval head and antennal tissues from early- and late-stage pupa to the adult of Bombus terrestris as suitable model organism among pollinators. We further performed Pearson correlation analyses on the gene expression profiles of the antennal transcriptome from larval head tissue to adult stages, exploring both positive and negative expression trends. The positively correlated coding genes were primarily enriched in sensory perception of chemical stimuli, ion transport, transmembrane transport processes and olfactory receptor activity. Negatively correlated genes were mainly enriched in organic substance biosynthesis and regulatory mechanisms underlying larval body patterning and the formation of juvenile antennal structures. As post-transcriptional regulators, miR-1000-5p, miR-13b-3p, miR-263-5p and miR-252-5p showed positive correlations, whereas miR-315-5p, miR-92b-3p, miR-137-3p, miR-11-3p and miR-10-3p exhibited negative correlations in antennal tissue. Notably, based on the inverse expression relationship, positively and negatively correlated microRNA (miRNA)-mRNA target pairs revealed that differentially expressed miRNAs predictively targeted genes involved in antennal development, shaping antennal structures and regulating antenna-specific functions. Our data serve as a foundation for understanding stage-specific antennal transcriptomes and large-scale comparative analysis of transcriptomes in different insects.

Bumblebees compensate for the adverse effects of sidewind during visually guided landings.

Flying animals often encounter winds during visually guided landings. However, how winds affect their flight control strategy during landing is unknown. Here, we investigated how sidewind affects the landing performance and sensorimotor control of foraging bumblebees (Bombus terrestris). We trained bumblebees to forage in a wind tunnel, and used high-speed stereoscopic videography to record 19,421 landing maneuvers in six sidewind speeds (0 to 3.4 m s-1), which correspond to winds encountered in nature. Bumblebees landed less often in higher windspeeds, but the landing durations from free flight were not increased by wind. By testing how bumblebees adjusted their landing control to compensate for adverse effects of sidewind on landing, we showed that the landing strategy in sidewind resembled that in still air, but with important adaptations. Bumblebees landing in a sidewind tended to drift downwind, which they controlled for by performing more hover maneuvers. Surprisingly, the increased hover prevalence did not increase the duration of free-flight landing maneuvers, as these bumblebees flew faster towards the landing platform outside the hover phases. Hence, by alternating these two flight modes along their flight path, free-flying bumblebees negated the adverse effects of high windspeeds on landing duration. Using control theory, we hypothesize that bumblebees achieve this by integrating a combination of direct aerodynamic feedback and a wind-mediated mechanosensory feedback control, with their vision-based sensorimotor control loop. The revealed landing strategy may be commonly used by insects landing in windy conditions, and may inspire the development of landing control strategies onboard autonomously flying robots.

Bumble bees do not avoid field-realistic but innocuous concentrations of cadmium and copper.

Bee populations are facing numerous stressors globally, including environmental pollution by trace metals and metalloids. Understanding whether bees can detect and avoid these pollutants in their food is pivotal, as avoidance abilities may mitigate their exposure to xenobiotics. While these pollutants are known to induce sublethal effects in bees, such as disrupting physiological mechanisms, their potential impacts on locomotive abilities, fat metabolism, and reproductive physiology remain poorly understood. In this study, utilising workers of the buff-tailed bumble bee and two prevalent trace metals, namely cadmium and copper, we aimed to address these knowledge gaps for field-realistic concentrations. Our findings reveal that workers did not reject field-realistic concentrations of cadmium and copper in sucrose solutions. Moreover, they did not reject lethal concentrations of cadmium, although they rejected lethal concentrations of copper. Additionally, we observed no significant effects of field-realistic concentrations of these metals on the walking and flying activities of workers, nor on their fat metabolism and reproductive physiology. Overall, our results suggest that bumble bees may not avoid cadmium and copper at environmental concentrations, but ingestion of these metals in natural settings may not adversely affect locomotive abilities, fat metabolism, or reproductive physiology. However, given the conservative nature of our study, we still recommend future research to employ higher concentrations over longer durations to mimic conditions in heavily polluted areas (i.e., mine surrounding). Furthermore, investigations should ascertain whether field-realistic concentrations of metals exert no impact on bee larvae.

Costs of reproduction are present but latent in eusocial bumblebee queens.

BACKGROUND: The standard evolutionary theory of ageing proposes that ageing occurs because of a trade-off between reproduction and longevity. Eusocial insect queens exhibit positive fecundity-longevity associations and so have been suggested to be counter-examples through not expressing costs of reproduction and through remodelling conserved genetic and endocrine networks regulating ageing and reproduction. If so, eusocial evolution from solitary ancestors with negative fecundity-longevity associations must have involved a stage at which costs of reproduction were suppressed and fecundity and longevity became positively associated. Using the bumblebee (Bombus terrestris), we experimentally tested whether queens in annual eusocial insects at an intermediate level of eusocial complexity experience costs of reproduction, and, using mRNA-seq, the extent to which they exhibit a remodelling of relevant genetic and endocrine networks. Specifically, we tested whether costs of reproduction are present but latent, or whether a remodelling of relevant genetic and endocrine networks has already occurred allowing queens to reproduce without costs. RESULTS: We experimentally increased queens' costs of reproduction by removing their eggs, which caused queens to increase their egg-laying rate. Treatment queens had significantly reduced longevity relative to control queens whose egg-laying rate was not increased. Reduced longevity in treatment queens was not caused by increased worker-to-queen aggression or by increased overall activity in queens. In addition, treatment and control queens differed in age-related gene expression based on mRNA-seq in both their overall expression profiles and the expression of ageing-related genes. Remarkably, these differences appeared to occur principally with respect to relative age, not chronological age. CONCLUSIONS: This study represents the first simultaneously phenotypic and transcriptomic experimental test for a longevity cost of reproduction in eusocial insect queens. The results support the occurrence of costs of reproduction in annual eusocial insects of intermediate social complexity and suggest that reproductive costs are present but latent in queens of such species, i.e. that these queens exhibit condition-dependent positive fecundity-longevity associations. They also raise the possibility that a partial remodelling of genetic and endocrine networks underpinning ageing may have occurred in intermediately eusocial species such that, in unmanipulated conditions, age-related gene expression depends more on chronological than relative age.

Walking bumblebees see faster.

The behavioural state of animals has profound effects on neuronal information processing. Locomotion changes the response properties of visual interneurons in the insect brain, but it is still unknown if it also alters the response properties of photoreceptors. Photoreceptor responses become faster at higher temperatures. It has therefore been suggested that thermoregulation in insects could improve temporal resolution in vision, but direct evidence for this idea has so far been missing. Here, we compared electroretinograms from the compound eyes of tethered bumblebees that were either sitting or walking on an air-supported ball. We found that the visual processing speed strongly increased when the bumblebees were walking. By monitoring the eye temperature during recording, we saw that the increase in response speed was in synchrony with a rise in eye temperature. By artificially heating the head, we show that the walking-induced temperature increase of the visual system is sufficient to explain the rise in processing speed. We also show that walking accelerates the visual system to the equivalent of a 14-fold increase in light intensity. We conclude that the walking-induced rise in temperature accelerates the processing of visual information-an ideal strategy to process the increased information flow during locomotion.

Limited introgression from non-native commercial strains and signatures of adaptation in the key pollinator Bombus terrestris.

Insect pollination is fundamental for natural ecosystems and agricultural crops. The bumblebee species Bombus terrestris has become a popular choice for commercial crop pollination worldwide due to its effectiveness and ease of mass rearing. Bumblebee colonies are mass produced for the pollination of more than 20 crops and imported into over 50 countries including countries outside their native ranges, and the risk of invasion by commercial non-native bumblebees is considered an emerging issue for global conservation and biological diversity. Here, we use genome-wide data from seven wild populations close to and far from farms using commercial colonies, as well as commercial populations, to investigate the implications of utilizing commercial bumblebee subspecies in the UK. We find evidence for generally low levels of introgression between commercial and wild bees, with higher admixture proportions in the bees occurring close to farms. We identify genomic regions putatively involved in local and global adaptation, and genes in locally adaptive regions were found to be enriched for functions related to taste receptor activity, oxidoreductase activity, fatty acid and lipid biosynthetic processes. Despite more than 30 years of bumblebee colony importation into the UK, we observe low impact on the genetic integrity of local B. terrestris populations, but we highlight that even limited introgression might negatively affect locally adapted populations.

Expression of subunits of an insecticide target receptor varies across tissues, life stages, castes, and species of social bees.

Global losses of insects jeopardize ecosystem stability and crop pollination. Robust evidence indicates that insecticides have contributed to these losses. Notably, insecticides targeting nicotinic acetylcholine receptors (nAChRs) have neurotoxic effects on beneficial insects. Because each nAChR consists of five subunits, the alternative arrangements of subunits could create a multitude of receptors differing in structure and function. Therefore, understanding whether the use of subunits varies is essential for evaluating and predicting the effects of insecticides targeting such receptors. To better understand how the use and composition of nAChRs differ within and between insect pollinators, we analysed RNA-seq gene expression data from tissues and castes of Apis mellifera honey bees and life stages and castes of the Bombus terrestris bumble bees. We reveal that all analysed tissues express nAChRs and that relative expression levels of nAChR subunits vary widely across almost all comparisons. Our work thus shows fine-tuned spatial and temporal expression of nAChRs. Given that coexpression of subunits underpins the compositional diversity of functional receptors and that the affinities of insecticides depend on nAChR composition, our findings provide a likely mechanism for the various damaging effects of nAChR-targeting insecticides on insects. Furthermore, our results indicate that the appraisal of insecticide risks should carefully consider variation in molecular targets.

Sterol and lipid metabolism in bees.

BACKGROUND: Bees provide essential pollination services for many food crops and are critical in supporting wild plant diversity. However, the dietary landscape of pollen food sources for social and solitary bees has changed because of agricultural intensification and habitat loss. For this reason, understanding the basic nutrient metabolism and meeting the nutritional needs of bees is becoming an urgent requirement for agriculture and conservation. We know that pollen is the principal source of dietary fat and sterols for pollinators, but a precise understanding of what the essential nutrients are and how much is needed is not yet clear. Sterols are key for producing the hormones that control development and may be present in cell membranes, where fatty-acid-containing species are important structural and signalling molecules (phospholipids) or to supply, store and distribute energy (glycerides). AIM OF THE REVIEW: In this critical review, we examine the current general understanding of sterol and lipid metabolism of social and solitary bees from a variety of literature sources and discuss implications for bee health. KEY SCIENTIFIC CONCEPTS OF REVIEW: We found that while eusocial bees are resilient to some dietary variation in sterol supply the scope for this is limited. The evidence of both de novo lipogenesis and a dietary need for particular fatty acids (FAs) shows that FA metabolism in insects is analogous to mammals but with distinct features. Bees rely on their dietary intake for essential sterols and lipids in a way that is dependent upon pollen availability.

Contrasting effects of fungicide and herbicide active ingredients and their formulations on bumblebee learning and behaviour.

Fungicides and herbicides are two of the most heavily applied pesticide classes in the world, but receive little research attention with regards to their potential impacts on bees. As they are not designed to target insects, the mechanisms behind potential impacts of these pesticides are unclear. It is therefore important to understand their influence at a range of levels, including sublethal impacts on behaviours such as learning. We used the proboscis extension reflex (PER) paradigm to assess how the herbicide glyphosate and the fungicide prothioconazole affect bumblebee olfactory learning. We also assessed responsiveness, and compared the impacts of these active ingredients and their respective commercial formulations (Roundup Biactive and Proline). We found that learning was not impaired by either formulation but, of the bees that displayed evidence of learning, exposure to prothioconazole active ingredient increased learning level in some situations, while exposure to glyphosate active ingredient resulted in bumblebees being less likely to respond to antennal stimulation with sucrose. Our data suggest that fungicides and herbicides may not negatively impact olfactory learning ability when bumblebees are exposed orally to field-realistic doses in a lab setting, but that glyphosate has the potential to cause changes in responsiveness in bees. As we found impacts of active ingredients and not commercial formulations, this suggests that co-formulants may modify impacts of active ingredients in the products tested on olfactory learning without being toxic themselves. More research is needed to understand the mechanisms behind potential impacts of fungicides and herbicides on bees, and to evaluate the implications of behavioural changes caused by glyphosate and prothioconazole for bumblebee fitness.

Elevated developmental temperatures impact the size and allometry of morphological traits of the bumblebee Bombus terrestris.

The impact of global warming on wild bee decline threatens the pollination services they provide. Exposure to temperatures above optimal during development is known to reduce adult body size but how it affects the development and scaling of body parts remains unclear. In bees, a reduction in body size and/or a reduction in body parts, such as the antennae, tongue and wings, and how they scale with body size (i.e. their allometry) could severely affect their fitness. To date, it remains unclear how temperature affects body size and the scaling of morphological traits in bees. To address this knowledge gap, we exposed both males and workers of Bombus terrestris to elevated temperature during development and assessed the effects on (i) the size of morphological traits and (ii) the allometry between these traits. Colonies were exposed to optimal (25°C) or stressful (33°C) temperatures. We then measured the body size, wing size, antenna and tongue length, as well as the allometry between these traits. We found that workers were smaller and the antennae of both castes were reduced at the higher temperature. However, tongue length and wing size were not affected by developmental temperature. The allometric scaling of the tongue was also affected by developmental temperature. Smaller body size and antennae could impair both individual and colony fitness, by affecting foraging efficiency and, consequently, colony development. Our results encourage further exploration of how the temperature-induced changes in morphology affect functional traits and pollination efficiency.

Perception, regulation, and fitness effects of pollen phytosterols in the bumble bee, Bombus terrestris.

PREMISE: Many flowering plants depend on insects for pollination and thus attract pollinators by offering rewards, mostly nectar and pollen. Bee pollinators rely on pollen as their main nutrient source. Pollen provides all essential micro- and macronutrients including substances that cannot be synthesized by bees themselves, such as sterols, which bees need for processes such as hormone production. Variations in sterol concentrations may consequently affect bee health and reproductive fitness. We therefore hypothesized that (1) these variations in pollen sterols affect longevity and reproduction in bumble bees and (2) can thus be perceived via the bees' antennae before consumption. METHODS: We studied the effect of sterols on longevity and reproduction of Bombus terrestris workers in feeding experiments and investigated sterol perception using chemotactile proboscis extension response (PER) conditioning. RESULTS: Workers could perceive several sterols (cholesterol, cholestenone, desmosterol, stigmasterol, ß-sitosterol) via their antennae but not differentiate between them. However, when sterols were presented in pollen, and not as a single compound, the bees were unable to differentiate between pollen differing in sterol content. Additionally, different sterol concentrations in pollen neither affected pollen consumption nor brood development or worker longevity. CONCLUSIONS: Since we used both natural concentrations and concentrations higher than those found in pollen, our results indicate that bumble bees may not need to pay specific attention to pollen sterol content beyond a specific threshold. Naturally encountered concentrations might fully support their sterol requirements and higher concentrations do not seem to have negative effects.

Morphological Variation in Bumblebees (Bombus terrestris) (Hymenoptera: Apidae) After Three Decades of an Island Invasion.

Introduced social insects can be highly invasive outside of their native range. Around the world, the introduction and establishment of the eusocial bumblebee Bombus terrestris (L. 1758) (Hymenoptera: Apidae) has negatively impacted native pollinators and ecosystems. Understanding how morphological variation is linked to environmental variation across invasive ranges can indicate how rapidly species may be diverging or adapting across novel ranges and may assist with predicting future establishment and spread. Here we investigate whether B. terrestris shows morphological variation related to environmental variation across the island of Tasmania (Australia) where it was introduced three decades ago. We collected 169 workers from 16 sites across Tasmania and related relative abundance and morphology to landscape-wide climate, land use, and vegetation structure. We found weak morphological divergence related to environmental conditions across Tasmania. Body size of B. terrestris was positively associated with the percentage of urban land cover, a relationship largely driven by a single site, possibly reflecting high resource availability in urban areas. Proboscis length showed a significant negative relationship with the percentage of pasture. Wing loading and local abundance were not related to the environmental conditions within sites. Our results reflect the highly adaptable nature of B. terrestris and its ability to thrive in different environments, which may have facilitated the bumblebee's successful invasion across Tasmania.

Untargeted Lipidomics Analysis Unravels the Different Metabolites in the Fat Body of Mated Bumblebee (Bombus terrestris) Queens.

The fat body has important functions in energy, fertility, and immunity. In female insects, mating stimulates physiological, behavioral, and gene expression changes. However, it remains unclear whether the metabolites in the fat body are affected after the bumblebee (Bombus terrestris) queen mates. Here, the ultrastructure and lipid metabolites in fat body of mated queens were compared with those of virgins. The fat body weight of mated bumblebee queens was significantly increased, and the adipocytes were filled with lipid droplets. Using LC-MS/MS-based untargeted lipidomics, 949 and 748 differential metabolites were identified in the fat body of virgin and mated bumblebee queens, respectively, in positive and negative ion modes. Most lipid metabolites were decreased, especially some biomembrane components. In order to explore the relationship between the structures of lipid droplets and metabolite accumulation, transmission electron microscopy and fluorescence microscopy were used to observe the fat body ultrastructure. The size/area of lipid droplets was larger, and the fusion of lipid droplets was increased in the mated queen's fat body. These enlarged lipid droplets may store more energy and nutrients. The observed differences in lipid metabolites in the fat body of queens contribute to understanding the regulatory network of bumblebees post mating.

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.

Comparative 4D Label-Free Quantitative Proteomic Analysis of Bombus terrestris Provides Insights into Proteins and Processes Associated with Diapause.

Diapause, an adaptative strategy for survival under harsh conditions, is a dynamic multi-stage process. Bombus terrestris, an important agricultural pollinator, is declining in the wild, but artificial breeding is possible by imitating natural conditions. Mated queen bees enter reproductive diapause in winter and recover in spring, but the regulatory mechanisms remain unclear. Herein, we conducted a comparative 4D label-free proteomic analysis of queen bees during artificial breeding at seven timepoints, including pre-diapause, diapause, and post-diapause stages. Through bioinformatics analysis of proteomic and detection of substance content changes, our results found that, during pre-diapause stages, queen bees had active mitochondria with high levels of oxidative phosphorylation, high body weight, and glycogen and TAG content, all of which support energy consumption during subsequent diapause. During diapause stages, body weight and water content were decreased but glycerol increased, contributing to cold resistance. Dopamine content, immune defense, and protein phosphorylation were elevated, while fat metabolism, protein export, cell communication, signal transduction, and hydrolase activity decreased. Following diapause termination, JH titer, water, fatty acid, and pyruvate levels increased, catabolism, synaptic transmission, and insulin signaling were stimulated, ribosome and cell cycle proteins were upregulated, and cell proliferation was accelerated. Meanwhile, TAG and glycogen content decreased, and ovaries gradually developed. These findings illuminate changes occurring in queen bees at different diapause stages during commercial production.

Field-realistic concentrations of a neonicotinoid insecticide influence socially regulated brood development in a bumblebee.

The systemic neonicotinoid insecticides are considered as one of the key culprits contributing to ongoing declines in pollinator health and abundance. Bumblebees are among the most important pollinators of temperate zone plants, making their susceptibility to neonicotinoid exposure of great concern. We report that bumblebee (Bombus terrestris) colonies exposed to field-realistic concentrations of the commonly used neonicotinoid Imidacloprid grew slower, consumed less food, and produced fewer workers, males and gynes, but unexpectedly produced larger workers compared to control colonies. Behavioural observations show that queens in pesticide-treated colonies spend more time inactive and less time caring for the brood. We suggest that the observed effects on brood body size are driven by a decreased queen ability to manipulate the larva developmental programme. These findings reveal an intricate and previously unknown effect of insecticides on the social interactions controlling brood development in social insect colonies. Insecticide influences on the social mechanisms regulating larval development are potentially detrimental for bumblebees, in which body size strongly influences both caste differentiation and the division of labour among workers, two organization principles of insect societies.