DNA methylation extends lifespan in the bumblebee Bombus terrestris.

Epigenetic alterations are a primary hallmark of ageing. In mammals, age-related epigenetic changes alter gene expression profiles, disrupt cellular homeostasis and physiological functions and, therefore, promote ageing. It remains unclear whether ageing is also driven by epigenetic mechanisms in invertebrates. Here, we used a pharmacological hypomethylating agent (RG108) to evaluate the effects of DNA methylation (DNAme) on lifespan in an insect-the bumblebee Bombus terrestris. RG108 extended mean lifespan by 43% and induced the differential methylation of genes involved in hallmarks of ageing, including DNA damage repair and chromatin organization. Furthermore, the longevity gene sirt1 was overexpressed following the treatment. Functional experiments demonstrated that SIRT1 protein activity was positively associated with lifespan. Overall, our study indicates that epigenetic mechanisms are conserved regulators of lifespan in both vertebrates and invertebrates and provides new insights into how DNAme is involved in the ageing process in insects.

Microrheology of haemolymph plasma of the bumblebee Bombus terrestris.

Viscosity, which impacts the rate of haemolymph circulation and heat transfer, is one of the transport properties that affects the performance of an insect. Measuring the viscosity of insect fluids is challenging because of the small amount available per specimen. Using particle tracking microrheology, which is well suited to characterise the rheology of the fluid part of the haemolymph, we studied the plasma viscosity in the bumblebee Bombus terrestris. In a sealed geometry, the viscosity exhibits an Arrhenius dependence with temperature, with an activation energy comparable to that previously estimated in hornworm larvae. In an open to air geometry, it increases by 4-5 orders of magnitude during evaporation. Evaporation times are temperature dependent and longer than typical insect haemolymph coagulation times. Unlike standard bulk rheology, microrheology can be applied to even smaller insects, paving the way to characterise biological fluids such as pheromones, pad secretions or cuticular layers.

Targeted Analysis of HSP70 Isoforms in Human Spermatozoa in the Context of Capacitation and Motility.

HSP70s constitute a family of chaperones, some isoforms of which appear to play a role in sperm function. Notably, global proteomic studies analyzing proteins deregulated in asthenozoospermia, a main cause of male infertility characterized by low sperm motility, showed the dysregulation of some HSP70 isoforms. However, to date, no clear trend has been established since the variations in the abundance of HSP70 isoforms differed between studies. The HSPA2 isoform has been reported to play a key role in fertilization, but its dysregulation and possible relocation during capacitation, a maturation process making the spermatozoon capable of fertilizing an oocyte, is debated in the literature. The aim of the present study was to investigate the fate of all sperm HSP70 isoforms during capacitation and in relation to sperm motility. Using Multiple-Reaction Monitoring (MRM) mass spectrometry, we showed that the relative abundance of all detected isoforms was stable between non-capacitated and capacitated spermatozoa. Immunofluorescence using two different antibodies also demonstrated the stability of HSP70 isoform localization during capacitation. We also investigated spermatozoa purified from 20 sperm samples displaying various levels of total and progressive sperm motility. We showed that the abundance of HSP70 isoforms is not correlated to sperm total or progressive motility.

Global patterns in bumble bee pollen collection show phylogenetic conservation of diet.

Bumble bees (Bombus) are a group of eusocial bees with a strongly generalised feeding pattern, collecting pollen from many different botanical families. Though predominantly generalists, some bumble bee species seem to have restricted dietary choices. It is unclear whether restricted diets in bumble bees are inherent or a function of local conditions due to a lack of data for many species across different regions. The objective of this study was to determine whether bumble bee species displayed specific patterns of pollen collection, and whether patterns were influenced by phylogenetic relatedness or tongue length, a trait known to be associated with structuring floral visitation. Bumble bee pollen collection patterns were quantified from 4,132 pollen loads taken from 58 bumble bee species, representing 24% of the pollen-collecting diversity of this genus. Phylogenetic trait mapping showed a conserved pattern of dietary dissimilarity across species, but not for dietary breadth. Dietary dissimilarity was driven by collection of Fabaceae, with the most similar species collecting around 50%-60% of their diet from this botanical family. The proportion of the diet collected from Fabaceae also showed a conserved phylogenetic signal. Greater collection of Fabaceae was associated with longer tongue lengths, with shorter tongued species focusing on alternative botanical families. However, this result was largely driven by phylogenetic relatedness, not tongue length per se. These results demonstrate that, though generalists, bumble bees are still subject to dietary restrictions that constrain their foraging choices. These dietary constraints have implications for their persistence should their core resources decline in abundance.

Global effects of extreme temperatures on wild bumblebees.

Climate plays a key role in shaping population trends and determining the geographic distribution of species because of limits in species' thermal tolerance. An evaluation of species tolerance to temperature change can therefore help predict their potential spatial shifts and population trends triggered by ongoing global warming. We assessed inter- and intraspecific variations in heat resistance in relation to body mass, local mean temperatures, and evolutionary relationships in 39 bumblebee species, a major group of pollinators in temperate and cold ecosystems, across 3 continents, 6 biomes, and 20 regions (2386 male specimens). Based on experimental bioassays, we measured the time before heat stupor of bumblebee males at a heatwave temperature of 40 °C. Interspecific variability was significant, in contrast to interpopulational variability, which was consistent with heat resistance being a species-specific trait. Moreover, cold-adapted species are much more sensitive to heat stress than temperate and Mediterranean species. Relative to their sensitivity to extreme temperatures, our results help explain recent population declines and range shifts in bumblebees following climate change.

Efectos Globales de las Temperaturas Extremas sobre Abejorros Silvestres Resumen El clima juega un papel importante en la configuración de las tendencias poblacionales y en la determinación de la distribución geográfica de las especies debido a los límites de la tolerancia térmica y al agua que tiene cada especie. Por lo tanto, una evaluación de la tolerancia de las especies al cambio térmico puede ayudar a predecir los potenciales cambios espaciales y las tendencias poblacionales detonadas por el calentamiento global en curso. Evaluamos las variaciones inter- e intraespecíficas de la resistencia al calor en relación con la masa corporal, temperaturas locales promedio y las relaciones evolutivas para 39 especies de abejorros, un grupo primordial de polinizadores en ecosistemas templados y fríos, en tres continentes, seis biomas y 20 regiones (2,386 especímenes machos). Con base en bioanálisis experimentales, medimos el tiempo previo al letargo por calor de los abejorros machos a temperaturas de 40 °C, típicas de una ola de calor. La variabilidad interespecífica fue significativa, en contraste con la variabilidad interpoblacional, lo cual fue consistente con el hecho de que la resistencia al calor sea una característica específica de la especie. Además, las especies adaptadas al frío son mucho más sensibles al estrés por calor que las especies de clima templado o Mediterráneo. En relación con la sensibilidad a las temperaturas extremas, nuestros resultados ayudan a explicar las recientes declinaciones poblacionales y cambios en la distribución de los abejorros posteriores al cambio climático.

Influence of Risk Factors for Male Infertility on Sperm Protein Composition.

Male infertility is a common health problem that can be influenced by a host of lifestyle risk factors such as environment, nutrition, smoking, stress, and endocrine disruptors. These effects have been largely demonstrated on sperm parameters (e.g., motility, numeration, vitality, DNA integrity). In addition, several studies showed the deregulation of sperm proteins in relation to some of these factors. This review inventories the literature related to the identification of sperm proteins showing abundance variations in response to the four risk factors for male infertility that are the most investigated in this context: obesity, diabetes, tobacco smoking, and exposure to bisphenol-A (BPA). First, we provide an overview of the techniques used to identify deregulated proteins. Then, we summarise the main results obtained in the different studies and provide a compiled list of deregulated proteins in relation to each risk factor. Gene ontology analysis of these deregulated proteins shows that oxidative stress and immune and inflammatory responses are common mechanisms involved in sperm alterations encountered in relation to the risk factors.

Shift in size of bumblebee queens over the last century.

Species can respond differently when facing environmental changes, such as by shifting their geographical ranges or through plastic or adaptive modifications to new environmental conditions. Phenotypic modifications related to environmental factors have been mainly explored along latitudinal gradients, but they are relatively understudied through time despite their importance for key ecological interactions. Here we hypothesize that the average bumblebee queen body size has changed in Belgium during the last century. Based on historical and contemporary databases, we first tested if queen body sizes changed during the last century at the intraspecific level among four common bumblebee species and if it could be linked to global warming and/or habitat fragmentation as well as by the replacement by individuals from new populations. Then, we assessed body size changes at the community level, among 22 species, taking into account species population trends (i.e. increasing, stable or decreasing relative abundance). Our results show that the average queen body size of all four bumblebee species increased over the last century. This size increase was significantly correlated to global warming and habitat fragmentation, but not explained by changes in the population genetic structure (i.e. colonization). At the community level, species with stable or increasing relative abundance tend to be larger than declining species. Contrary to theoretical expectations from Bergmann's rule (i.e. increasing body size in colder climates), temperature does not seem to be the main driver of bumblebee body size during the last century as we observed the opposite body size trend. However, agricultural intensification and habitat fragmentation could be alternative mechanisms that shape body size clines. This study stresses the importance of considering alternative global change factors when assessing body size change.

Variability in Sexual Pheromones Questions their Role in Bumblebee Pre-Mating Recognition System.

Sex-specific chemical secretions have been widely used as diagnostic characters in chemotaxonomy. The taxonomically confused group of bumblebees has reaped the benefit of this approach through the analyses of cephalic labial gland secretions (CLGS). Most of currently available CLGS descriptions concern species from the West-Palearctic region but few from the New World. Here, the CLGS of four East-Palearctic species Bombus deuteronymus, B. filchnerae, B. humilis, and B. exil (subgenus Thoracobombus) are analysed. Our results show high levels of variability in the major compounds in B. exil. In contrast, we describe a low differentiation in CLGS compounds between B. filchnerae and its phylogenetically closely related taxon B. muscorum. Moreover, the chemical profiles of B. filchnerae and B. muscorum are characterized by low concentrations of the C16 component, which is found in higher concentrations in the other Thoracobombus species. This raises the possibility that courtship behavior as well as environmental constraints could affect the role of the bumblebee males' CLGS.

First Chemical Analysis and Characterization of the Male Species-Specific Cephalic Labial-Gland Secretions of South American Bumblebees.

The evolution of signals and reproductive traits involved in the pre-mating recognition has been in focus of abundant research in several model species, such as bumblebees (genus Bombus). However, the most-studied bumblebee reproductive trait, the male cephalic labial gland secretions (CLGS), remains unknown among bumblebee species from South America. In this study, the CLGS of five South American bumblebees of the subgenera Thoracobombus (Bombus excellens and B. atratus) and Cullumanobombus (B. rubicundus, B. hortulanus, and B. melaleucus) were investigated, by comparing the chemical compositions of their secretions to those of closely related European species. The results showed an obvious interspecific differentiation in both subgenera. The interspecific differentiation among the species of the Thoracobombus subgenus involved different compounds present at high contents (main compounds), while those of the Cullumanobombus subgenus shared the same main components. This suggests that among the species of the Cullumanobombus subgenus, the differentiation in minor components could lead to species discrimination.

Proteomic differences in seminal fluid of social insects whose sperm differ in heat tolerance.

In the coming years, climate change is likely to increase the frequency and intensity of heatwaves. In many organisms, heat stress provokes physiological perturbations and can lead to decreased male fertility. Bumblebees are endo-heterothermic but display interspecific differences in thermotolerance that could have conservation implications. For the species of concern Bombus magnus, exposure to high temperatures can severely reduce sperm quality and, consequently, reproductive success. Such is not the case for B. terrestris, a ubiquitous species. To decipher the mechanisms at play, we characterized the seminal fluid proteomes of the two species. We quantified 1121 proteins, of which 522 were differentially expressed between B. terrestris and B. magnus. Several proteins with protective functions, such as proteases, antioxidant proteins and various heat-shock proteins, were present at higher levels in B. terrestris than in B. magnus under both control and heat-stress conditions. The same was true for proteins involved in cellular homeostasis, immunity, lipid/sugar metabolism and thermotolerance. Furthermore, proteins involved in the capture and elimination of reactive oxygen species also occurred at much high levels in B. terrestris. Overall, these results clearly indicate differences in the seminal proteome of the more thermotolerant B. terrestris versus B. magnus. The differences may contribute to explaining interspecific differences in sperm survival.

The ribosome inhibitor chloramphenicol induces motility deficits in human spermatozoa: A proteomic approach identifies potentially involved proteins.

Mature spermatozoa are almost completely devoid of cytoplasm; as such it has long been believed that they do not contain ribosomes and are therefore not capable of synthesising proteins. However, since the 1950s, various studies have shown translational activity within spermatozoa, particularly during their in vitro capacitation. But the type of ribosomes involved (cytoplasmic or mitochondrial) is still debated. Here, we investigate the presence and activity of the two types of ribosomes in mature human spermatozoa. By targeting ribosomal RNAs and proteins, we show that both types of ribosomes are localized in the midpiece as well as in the neck and the base of the head of the spermatozoa. We assessed the impact of cycloheximide (CHX) and chloramphenicol (CP), inhibitors of cytoplasmic and mitochondrial ribosomes, respectively, on different sperm parameters. Neither CHX, nor CP impacted sperm vitality, mitochondrial activity (measured through the ATP content), or capacitation (measured through the content in phosphotyrosines). However, increasing CP concentrations induced a decrease in total and progressive motilities as well as on some kinematic parameters while no effect was observed with CHX. A quantitative proteomic analysis was performed by mass spectrometry in SWATH mode to compare the proteomes of spermatozoa capacitated in the absence or presence of the two ribosome inhibitors. Among the ∼700 proteins identified in the different tested conditions, 3, 3 and 25 proteins presented a modified abundance in the presence of 1 and 2 mg/ml of CHX, and 1 mg/ml of CP, respectively. The observed abundance variations of some CP-down regulated proteins were validated using Multiple-Reaction Monitoring (MRM). Taken together, our results are in favor of an activity of mitochondrial ribosomes. Their inhibition by CP results in a decrease in the abundance of several proteins, at least FUNDC2 and QRICH2, and consequently induces sperm motility deficits.

Sexual attraction: a review of bumblebee male pheromones.

Males of many bumblebee species exhibit a conspicuous pre-mating behavior with two distinct behavioral components: scent marking and patrol flying. The marking pheromone is produced by the cephalic part of the labial gland (CLG). As far as is known, the CLG secretion is species specific, and it usually consists of two types of compounds: (i) straight-chain aliphatic alcohols, aldehydes or esters, and (ii) acyclic mono-, sesqui- and diterpenes (alcohols or acetates). Here, we summarize data from the literature reporting chemical composition of the CLG secretions of more than 80 bumblebee species. Similarities and differences within and between subgenera are discussed in the context of biosynthetic pathways and evolution.

Ensuring access to high-quality resources reduces the impacts of heat stress on bees.

Pollinators are experiencing declines globally, negatively affecting the reproduction of wild plants and crop production. Well-known drivers of these declines include climatic and nutritional stresses, such as a change of dietary resources due to the degradation of habitat quality. Understanding potential synergies between these two important drivers is needed to improve predictive models of the future effects of climate change on pollinator declines. Here, bumblebee colony bioassays were used to evaluate the interactive effects of heat stress, a reduction of dietary resource quality, and colony size. Using a total of 117 colonies, we applied a fully crossed experiment to test the effect of three dietary quality levels under three levels of heat stress with two colony sizes. Both nutritional and heat stress reduced colony development resulting in a lower investment in offspring production. Small colonies were much more sensitive to heat and nutritional stresses than large ones, possibly because a higher percentage of workers helps maintain social homeostasis. Strikingly, the effects of heat stress were far less pronounced for small colonies fed with suitable diets. Overall, our study suggests that landscape management actions that ensure access to high-quality resources could reduce the impacts of heat stress on bee decline.

The cephalic labial gland secretions of two socially parasitic bumblebees Bombus hyperboreus (Alpinobombus) and Bombus inexspectatus (Thoracobombus) question their inquiline strategy.

Social parasitic Hymenopterans have evolved morphological, chemical, and behavioral adaptations to overcome the sophisticated recognition and defense systems of their social host to invade host nests and exploit their worker force. In bumblebees, social parasitism appeared in at least 3 subgenera independently: in the subgenus Psithyrus consisting entirely of parasitic species, in the subgenus Alpinobombus with Bombus hyperboreus, and in the subgenus Thoracobombus with B. inexspectatus. Cuckoo bumblebee males utilize species-specific cephalic labial gland secretions for mating purposes that can impact their inquiline strategy. We performed cephalic labial gland secretions in B. hyperboreus, B. inexspectatus and their hosts. Males of both parasitic species exhibited high species specific levels of cephalic gland secretions, including different main compounds. Our results showed no chemical mimicry in the cephalic gland secretions between inquilines and their host and we did not identify the repellent compounds already known in other cuckoo bumblebees.

A protocol to assess insect resistance to heat waves, applied to bumblebees (Bombus Latreille, 1802).

Insect decline results from numerous interacting factors including climate change. One of the major phenomena related to climate change is the increase of the frequency of extreme events such as heat waves. Since heat waves are suspected to dramatically increase insect mortality, there is an urgent need to assess their potential impact. Here, we determined and compared the resistance to heat waves of insects under hyperthermic stress through their time before heat stupor (THS) when they are exposed to an extreme temperature (40°C). For this, we used a new experimental standardised device available in the field or in locations close to the field collecting sites. We applied this approach on different Arctic, Boreo-Alpine and Widespread bumblebee species in order to predict consequences of heat waves. Our results show a heat resistance gradient: the heat stress resistance of species with a centred arctic distribution is weaker than the heat resistance of the Boreo-Alpine species with a larger distribution which is itself lower than the heat stress resistance of the ubiquitous species.