Towards unbiased interpretations of interactive effects in ecotoxicological studies.

Ecotoxicological research has increasingly focused on the interactive effects of chemical mixtures on biological models, emphasising additive, synergistic, or antagonistic interactions. However, these combination studies often test chemicals at unique concentrations (e.g. x:y), limiting our understanding of the effects across the full spectrum of possible combinations. Evidence from human toxicology suggests that interactive effects among chemicals can vary significantly with total concentration (e.g. x:y vs. 2x:2y), their ratio (e.g. x:2y vs. 2x:y), and the magnitude of the tested effect (e.g. LC10vs. LC50). Our non-exhaustive review of studies on binary mixtures in bee ecotoxicology reveals that such parameters are frequently neglected. Of the 60 studies we examined, only two utilised multiple total concentrations and ratios, thus exploring a broad range of possible combinations. In contrast, 26 studies tested only a single concentration of each chemical, resulting in incomplete interpretations of the potential interactive effects. Other studies utilised various concentrations and/or ratios but failed to capture a broad spectrum of possible combinations. We also discuss potential discrepancies in interactive effects based on different metrics and exposure designs. We advocate for future ecotoxicological studies to investigate a wider spectrum of chemical combinations, including various concentrations and ratios, and to address different levels of effects.

Exposure to elevated temperature during development affects eclosion and morphology in the temperate Pieris napi butterfly (Lepidoptera: Pieridae).

Global warming has been identified as one of the main drivers of population decline in insect pollinators. One aspect of the insect life cycle that would be particularly sensitive to elevated temperatures is the developmental transition from larva to adult. Temperature-induced modifications to the development of body parts and sensory organs likely have functional consequences for adult behaviour. To date, we have little knowledge about the effect of sub-optimal temperature on the development and functional morphology of different body parts, particularly sensory organs, in ectothermic solitary pollinators such as butterflies. To address this knowledge gap, we exposed the pupae of the butterfly Pieris napi to either 23 °C or 32 °C and measured the subsequent effects on eclosion, body size and the development of the wings, proboscis, eyes and antennae. In comparison to individuals that developed at 23 °C, we found that exposure to 32 °C during the pupal stage increased mortality and decreased time to eclose. Furthermore, both female and male butterflies that developed at 32 °C were smaller and had shorter proboscides, while males had shorter antennae. In contrast, we found no significant effect of rearing temperature on wing and eye size or wing deformity. Our findings suggest that increasing global temperatures and its corresponding co-stressors, such as humidity, will impact the survival of butterflies by impairing eclosion and the proper development of body and sensory organs.

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.

Exposure to elevated temperature during development affects bumblebee foraging behavior.

Bee foraging behavior provides a pollination service that has both ecological and economic benefits. However, bee population decline could directly affect the efficiency of this interaction. Among the drivers of this decline, global warming has been implicated as an emerging threat but exactly how increasing temperatures affect bee foraging behavior remains unexplored. Here, we assessed how exposure to elevated temperatures during development affects the foraging behavior and morphology of workers from commercial and wild Bombus terrestris colonies. Workers reared at 33 °C had a higher visiting rate and shorter visiting time than those reared at 27°C. In addition, far fewer workers reared at 33 °C engaged in foraging activities and this is potentially related to the drastic reduction in the number of individuals produced in colonies exposed to 33 °C. The impact of elevated developmental temperature on wild colonies was even stronger as none of the workers from these colonies performed any foraging trips. We also found that rearing temperature affected wing size and shape. Our results provide the first evidence that colony temperature can have striking effects on bumblebee foraging behavior. Of particular importance is the drastic reduction in the number of workers performing foraging trips, and the total number of foraging trips made by workers reared in high temperatures. Further studies should explore if, ultimately, these observed effects of exposure to elevated temperature during development lead to a reduction in pollination efficiency.

Poison or Potion: Effects of Sunflower Phenolamides on Bumble Bees and Their Gut Parasite.

Specific floral resources may help bees to face environmental challenges such as parasite infection, as recently shown for sunflower pollen. Whereas this pollen diet is known to be unsuitable for the larval development of bumble bees, it has been shown to reduce the load of a trypanosomatid parasite (Crithidia bombi) in the bumble bee gut. Recent studies suggested it could be due to phenolamides, a group of compounds commonly found in flowering plants. We, therefore, decided to assess separately the impacts of sunflower pollen and its phenolamides on a bumble bee and its gut parasite. We fed Crithidia-infected and -uninfected microcolonies of Bombus terrestris either with a diet of willow pollen (control), a diet of sunflower pollen (natural diet) or a diet of willow pollen supplemented with sunflower phenolamides (supplemented diet). We measured several parameters at both microcolony (i.e., food collection, parasite load, brood development and stress responses) and individual (i.e., fat body content and phenotypic variation) levels. As expected, the natural diet had detrimental effects on bumble bees but surprisingly, we did not observe any reduction in parasite load, probably because of bee species-specific outcomes. The supplemented diet also induced detrimental effects but by contrast to our a priori hypothesis, it led to an increase in parasite load in infected microcolonies. We hypothesised that it could be due to physiological distress or gut microbiota alteration induced by phenolamide bioactivities. We further challenged the definition of medicinal effects and questioned the way to assess them in controlled conditions, underlining the necessity to clearly define the experimental framework in this research field.

Short-term exposure to heatwave-like temperatures affects learning and memory in bumblebees.

Global warming has been identified as a key driver of bee declines around the world. While it is clear that elevated temperatures during the spring and summer months-the principal activity period of many bee species-is a factor in this decline, exactly how temperature affects bee survival is unknown. In vertebrates, there is clear evidence that elevated ambient temperatures impair cognition but whether and how heat affects the cognitive abilities of invertebrates remains unclear. Cognitive skills in bees are essential for their survival as, to supply the hive with nutrition, workers must be able to learn and remember the location of the most rewarding floral resources. Here, we investigate whether temperature-related cognitive impairments could be a driver of bee declines by exploring the effect of short-term increases in ambient temperature on learning and memory. We found that, in comparison to bees that were tested at 25°C (a temperature that they would typically experience in summer), bees that were exposed to 32°C (a temperature that they will becoming increasingly exposed to during heatwave events) were significantly worse at forming an association between a coloured light and a sucrose reward and that their capacity to remember this association after just 1 h was abolished. This study provides novel experimental evidence that even just a few hours of exposure to heatwave-like temperatures can severely impair the cognitive performance of insects. Such temperature-induced cognitive deficits could play an important role in explaining recent and future bee population declines.

Resilience of bumblebee foraging behavior despite colony size reduction.

Foraging behavior is driven by diverse factors, notably life history traits. Foraging strategies are particularly complex among eusocial species such as bumblebees, because they depend primarily on the needs of the colony, rather than on individual's needs. Colony size, i.e. the number of workers in a colony vary a lot among eusocial insects. While a large colony can be adaptive, several drivers can strongly decrease colony size, like pesticides or high temperatures. In this study, we used the bumblebee Bombus terrestris to assess if workers adapted their foraging behavior to such rapid decreases in colony size. We conducted the foraging experiments with two plant species commonly used by bumblebees: Borago officinalis and Echium plantagineum. Several foraging parameters were measured: foraging time, number of foraging trips, number of workers foraging, handling time and visiting rate. Despite a drastic reduction in colony size, nearly all the foraging behavior parameters were unaffected by the colony size reduction. Colonies that were subject to a large decrease in workers instead displayed high resilience and behavioral plasticity by quickly increasing the proportion of foragers. Ultimately, further research should assess if this consistency in foraging behavior also allows bumblebee colonies to maintain both the efficiency of the resources collection and pollination.

Expanding insect pollinators in the Anthropocene.

Global changes are severely affecting pollinator insect communities worldwide, resulting in repeated patterns of species extirpations and extinctions. Whilst negative population trends within this functional group have understandably received much attention in recent decades, another facet of global changes has been overshadowed: species undergoing expansion. Here, we review the factors and traits that have allowed a fraction of the pollinating entomofauna to take advantage of global environmental change. Sufficient mobility, high resistance to acute heat stress, and inherent adaptation to warmer climates appear to be key traits that allow pollinators to persist and even expand in the face of climate change. An overall flexibility in dietary and nesting requirements is common in expanding species, although niche specialization can also drive expansion under specific contexts. The numerous consequences of wild and domesticated pollinator expansions, including competition for resources, pathogen spread, and hybridization with native wildlife, are also discussed. Overall, we show that the traits and factors involved in the success stories of expanding pollinators are mostly species specific and context dependent, rendering generalizations of 'winning traits' complicated. This work illustrates the increasing need to consider expansion and its numerous consequences as significant facets of global changes and encourages efforts to monitor the impacts of expanding insect pollinators, particularly exotic species, on natural ecosystems.

Long-term effects of global change on occupancy and flight period of wild bees in Belgium.

Global change affects species by modifying their abundance, spatial distribution, and activity period. The challenge is now to identify the respective drivers of those responses and to understand how those responses combine to affect species assemblages and ecosystem functioning. Here we correlate changes in occupancy and mean flight date of 205 wild bee species in Belgium with temporal changes in temperature trend and interannual variation, agricultural intensification, and urbanization. Over the last 70 years, bee occupancy decreased on average by 33%, most likely because of agricultural intensification, and flight period of bees advanced on average by 4 days, most likely because of interannual temperature changes. Those responses resulted in a synergistic effect because species which increased in occupancy tend to be those that have shifted their phenologies earlier in the season. This leads to an overall advancement and shortening of the pollination season by 9 and 15 days respectively, with lower species richness and abundance compared to historical pollinator assemblages, except at the early start of the season. Our results thus suggest a strong decline in pollination function and services.

Beyond the Decline of Wild Bees: Optimizing Conservation Measures and Bringing Together the Actors.

Wild bees are facing a global decline mostly induced by numerous human factors for the last decades. In parallel, public interest for their conservation increased considerably, namely through numerous scientific studies relayed in the media. In spite of this broad interest, a lack of knowledge and understanding of the subject is blatant and reveals a gap between awareness and understanding. While their decline is extensively studied, information on conservation measures is often scattered in the literature. We are now beyond the precautionary principle and experts are calling for effective actions to promote wild bee diversity and the enhancement of environment quality. In this review, we draw a general and up-to-date assessment of the conservation methods, as well as their efficiency and the current projects that try to fill the gaps and optimize the conservation measures. Targeting bees, we focused our attention on (i) the protection and restoration of wild bee habitats, (ii) the conservation measures in anthropogenic habitats, (iii) the implementation of human made tools, (iv) how to deal with invasive alien species, and finally (v) how to communicate efficiently and accurately. This review can be considered as a needed catalyst to implement concrete and qualitative conversation actions for bees.

Global warming and plant-pollinator mismatches.

The mutualism between plants and their pollinators provides globally important ecosystem services, but it is likely to be disrupted by global warming that can cause mismatches between both halves of this interaction. In this review, we summarise the available evidence on (i) spatial or (ii) phenological shifts of one or both of the actors of this mutualism. While the occurrence of future spatial mismatches is predominantly theoretical and based on predictive models, there is growing empirical evidence of phenological mismatches occurring at the present day. Mismatches may also occur when pollinators and their host plants are still found together. These mismatches can arise due to (iii) morphological modifications and (iv) disruptions to host attraction and foraging behaviours, and it is expected that these mismatches will lead to novel community assemblages. Overall plant-pollinator interactions seem to be resilient biological networks, particularly because generalist species can buffer these changes due to their plastic behaviour. However, we currently lack information on where and why spatial mismatches do occur and how they impact the fitness of plants and pollinators, in order to fully assess if adaptive evolutionary changes can keep pace with global warming predictions.

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.

Morphometric analysis of fossil bumble bees (Hymenoptera, Apidae, Bombini) reveals their taxonomic affinities.

Bumble bees (Bombus spp.) are a widespread corbiculate lineage (Apinae: Corbiculata: Bombini), mostly found among temperate and alpine ecosystems. Approximately 260 species have been recognized and grouped recently into a simplified system of 15 subgenera. Most of the species are nest-building and primitively eusocial. Species of Bombus have been more intensely studied than any other lineages of bees with the exception of the honey bees. However, most bumble bee fossils are poorly described and documented, making their placement relative to other Bombus uncertain. A large portion of the known and presumed bumble bee fossils were re-examined in an attempt to better understand their affinities with extant Bombini. The taxonomic affinities of fossil specimens were re-assessed based on morphological features and previous descriptions, and for 13 specimens based on geometric morphometrics of forewing shape. None of the specimens coming from Eocene and Oligocene deposits were assigned within the contemporary shape space of any subgenus of Bombus. It is shown that Calyptapis florissantensis Cockerell, 1906 (Eocene-Oligocene boundary, Florissant shale, Colorado, USA) and Oligobombus cuspidatus Antropov, 2014 (Late Eocene, Bembridge Marls) likely belong to stem-group Bombini. Bombus anacolus Zhang, 1994, B. dilectus Zhang, 1994, B. luianus Zhang, 1990 (Middle Miocene, Shanwang Formation), as well as B. vetustus Rasnitsyn & Michener, 1991 (Miocene, Botchi Formation) are considered as species inquirenda. In the Miocene, affinities of fossils with derived subgenera of Bombus s. l. increased, and some are included in the shape space of contemporary subgenera: Cullumanobombus (i.e., B. pristinus Unger, 1867, B. randeckensis Wappler & Engel, 2012, and B. trophonius Prokop, Dehon, Michez & Engel, 2017), Melanobombus (i.e., B. cerdanyensis Dehon, De Meulemeester & Engel, 2014), and Mendacibombus (i.e., B. beskonakensis (Nel & Petrulevicius, 2003), new combination), agreeing with previous estimates of diversification.

Stressful conditions reveal decrease in size, modification of shape but relatively stable asymmetry in bumblebee wings.

Human activities can generate a wide variety of direct and indirect effects on animals, which can manifest as environmental and genetic stressors. Several phenotypic markers have been proposed as indicators of these stressful conditions but have displayed contrasting results, depending, among others, on the phenotypic trait measured. Knowing the worldwide decline of multiple bumblebee species, it is important to understand these stressors and link them with the drivers of decline. We assessed the impact of several stressors (i.e. natural toxin-, parasite-, thermic- and inbreeding- stress) on both wing shape and size and their variability as well as their directional and fluctuating asymmetries. The total data set includes 650 individuals of Bombus terrestris (Hymenoptera: Apidae). Overall wing size and shape were affected by all the tested stressors. Except for the sinigrin (e.g. glucosinolate) stress, each stress implies a decrease of wing size. Size variance was affected by several stressors, contrary to shape variance that was affected by none of them. Although wing size directional and fluctuating asymmetries were significantly affected by sinigrin, parasites and high temperatures, neither directional nor fluctuating shape asymmetry was significantly affected by any tested stressor. Parasites and high temperatures led to the strongest phenotype modifications. Overall size and shape were the most sensitive morphological traits, which contrasts with the common view that fluctuating asymmetry is the major phenotypic marker of stress.

Distribution and predictors of wing shape and size variability in three sister species of solitary bees.

Morphological traits can be highly variable over time in a particular geographical area. Different selective pressures shape those traits, which is crucial in evolutionary biology. Among these traits, insect wing morphometry has already been widely used to describe phenotypic variability at the inter-specific level. On the contrary, fewer studies have focused on intra-specific wing morphometric variability. Yet, such investigations are relevant to study potential convergences of variation that could highlight micro-evolutionary processes. The recent sampling and sequencing of three solitary bees of the genus Melitta across their entire species range provides an excellent opportunity to jointly analyse genetic and morphometric variability. In the present study, we first aim to analyse the spatial distribution of the wing shape and centroid size (used as a proxy for body size) variability. Secondly, we aim to test different potential predictors of this variability at both the intra- and inter-population levels, which includes genetic variability, but also geographic locations and distances, elevation, annual mean temperature and precipitation. The comparison of spatial distribution of intra-population morphometric diversity does not reveal any convergent pattern between species, thus undermining the assumption of a potential local and selective adaptation at the population level. Regarding intra-specific wing shape differentiation, our results reveal that some tested predictors, such as geographic and genetic distances, are associated with a significant correlation for some species. However, none of these predictors are systematically identified for the three species as an important factor that could explain the intra-specific morphometric variability. As a conclusion, for the three solitary bee species and at the scale of this study, our results clearly tend to discard the assumption of the existence of a common pattern of intra-specific signal/structure within the intra-specific wing shape and body size variability.

Chemical reproductive traits of diploid Bombus terrestris males: Consequences on bumblebee conservation.

The current bumblebee decline leads to inbreeding in populations that fosters a loss of allelic diversity and diploid male production. As diploid males are viable and their offspring are sterile, bumblebee populations can quickly fall in a vortex of extinction. In this article, we investigate for the first time a potential premating mechanism through a major chemical reproductive trait (male cephalic labial gland secretions) that could prevent monandrous virgin queens from mating with diploid males. We focus our study on the cephalic labial gland secretions of diploid and haploid males of Bombus terrestris (L.). Contrary to initial expectations, our results do not show any significant differentiation of cephalic labial gland secretions between diploid and haploid specimens. Queens seem therefore to be unable to avoid mating with diploid males based on their compositions of cephalic labial gland secretions. This suggests that the vortex of extinction of diploid males could not be stopped through premating avoidance based on the cephalic labial gland secretions but other mechanisms could avoid mating between diploid males and queens.