Baptiste Martinet

Wildlife faces an increasing threat from extreme climatic events, such as heatwaves, which can have a severe impact on various species, including crucial pollinators like bumblebees. Bumblebees are cold‐adapted and heterothermic, possessing the ability to regulate their internal temperature. The impact of heat stress seems species specific in bumblebees. While most species are impacted, some bumblebee species manage to survive, potentially by employing physiological mechanisms, including the modulation of their protein profile (e.g. Heat Shock Proteins). However, there is limited understanding of how their protein profiles are associated with heat exposure. In this study, we examined the global variation in the protein profile of males from two bumblebee species sampled in the wild: the heat‐tolerant Bombus terrestris and the heat‐sensitive Bombus magnus . After subjecting them to heat stupor at 40°C in controlled condition, it was observed that nearly all B. terrestris survived the stress, while over 50% of B. magnus individuals succumbed to the heat exposure. Through off‐gel bottom‐up proteomics and LC–MS/MS analysis of the hemolymph proteome, we identified 164 proteins in both species with a large part of differentially expressed proteins after heat exposure. Additionally, quantitative analysis of fat bodies revealed that the relative mass was stable in B. terrestris , while it was significantly lower in B. magnus exposed to heat stress. Our data suggest that compared with B. magnus , B. terrestris displays a higher adaptability of its hemolymph proteome in response to heat stress. This adaptability could be a key factor contributing to the high physiological resistance of B. terrestris and its ability to adapt to new, stressful environments expected due to climate change. Understanding these mechanisms of protein regulation in bumblebees could provide valuable insights into their resilience and vulnerability facing environmental stresses.

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.

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.

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.

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.

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.