Nurse bees regulate the larval nutrition of developing workers (Apis mellifera) when feeding on various pollen types.

Nutrition has been identified as a key driver of colony health and productivity. Yet, in honey bees, relatively little is known about how the vast variety of natural pollen sources impact larval development. The impact of the nutritional quality of 4 naturally occurring pollen sources, of importance to the Western Australian beekeeping industry, was tested on honey bee (Apis mellifera L.) development. Bee packages consisting of 800 g of bees and a mated sister queen were assigned to 40 nucleus hives and randomly allocated to one of the 4 feed treatments (10 colonies each) of marri (Corymbia calophylla Lindl.), jarrah (Eucalyptus marginata Sm.), clover (Trifolium repens L.), and canola (Brassica napus L.) pollen. Emerging bees were collected once the first bees started hatching on the assigned feed sources. Newly emerging bees were weighed individually, and body composition was measured in batches according to the feed treatment groups. Food consumption was recorded for the duration of the experiment. Nurse bees successfully raised young adult workers from the larval stage until emergence when fed with one of 4 pollen patties with different nutritional qualities. There was no difference in the body composition or weight of emerging bees fed on the different pollen types. However, the body weight of bees increased over time, most likely related to colony size and structure. With the type of pollen patties having little impact on larval development, the availability of pollen may be more important than its composition, providing bees have access to all essential nutrients.

Larval Exposure to Parasitic Varroa destructor Mites Triggers Specific Immune Responses in Different Honey Bee Castes and Species.

Innate immune systems are key defenses of animals and particularly important in species that lack the sophisticated adaptive immune systems as found in vertebrates. Here, we were interested to quantify variation in innate immune responses of insects in hosts that differ in their parasite susceptibility. To do this, we studied immune responses in honey bees, which can host a remarkable number of different parasites, which are major contributors of declining bee health and colony losses. The most significant parasite of honey bees is the mite Varroa destructor, which has infested the majority of global honey bee populations, and its control remains a major challenge for beekeepers. However, a number of nonmanaged honey bees seem able to control Varroa infections, for example, the Eastern honey bee Apis ceranacerana or the African honey bee Apis mellifera scutellata. These bees therefore make interesting study subjects to identify underlaying resistance traits, for example, by comparing them to more susceptible bee genotypes such as Western honey bees (A. melliferaligustica). We conducted a series of interlinked experiments and started with behavioral assays to compare the attractiveness of bee larvae to mites using different honey bee genotypes and castes. We found that 6-day-old larvae are always most attractive to mites, independently of genotype or castes. In a next step, we compared volatile profiles of the most attractive larvae to test whether they could be used by mites for host selection. We found that the abundance of volatile compounds differed between larval ages, but we also found significant differences between genotypes and castes. To further study the expected underlaying physiological differences between potentially resistant and susceptible host larvae, we compared the larval hemolymph proteomes of the three honey bee genotypes and two castes in response to mite exposure. We identified consistent upregulation of immune and stress-related genes in Varroa-exposed larvae, which differed between genotypes and castes. Tolerant honey bee castes and genotypes were characterized by stronger or more distinct immune esponses. In summary, we provide first insights into the complex involvement of the innate immune system of tolerant honey bees against mite infestations, which could be used for future breeding purposes.

The emergence of the brain non-CpG methylation system in vertebrates.

Mammalian brains feature exceptionally high levels of non-CpG DNA methylation alongside the canonical form of CpG methylation. Non-CpG methylation plays a critical regulatory role in cognitive function, which is mediated by the binding of MeCP2, the transcriptional regulator that when mutated causes Rett syndrome. However, it is unclear whether the non-CpG neural methylation system is restricted to mammalian species with complex cognitive abilities or has deeper evolutionary origins. To test this, we investigated brain DNA methylation across 12 distantly related animal lineages, revealing that non-CpG methylation is restricted to vertebrates. We discovered that in vertebrates, non-CpG methylation is enriched within a highly conserved set of developmental genes transcriptionally repressed in adult brains, indicating that it demarcates a deeply conserved regulatory program. We also found that the writer of non-CpG methylation, DNMT3A, and the reader, MeCP2, originated at the onset of vertebrates as a result of the ancestral vertebrate whole-genome duplication. Together, we demonstrate how this novel layer of epigenetic information assembled at the root of vertebrates and gained new regulatory roles independent of the ancestral form of the canonical CpG methylation. This suggests that the emergence of non-CpG methylation may have fostered the evolution of sophisticated cognitive abilities found in the vertebrate lineage.

Honeybee venom and melittin suppress growth factor receptor activation in HER2-enriched and triple-negative breast cancer.

Despite decades of study, the molecular mechanisms and selectivity of the biomolecular components of honeybee (Apis mellifera) venom as anticancer agents remain largely unknown. Here, we demonstrate that honeybee venom and its major component melittin potently induce cell death, particularly in the aggressive triple-negative and HER2-enriched breast cancer subtypes. Honeybee venom and melittin suppress the activation of EGFR and HER2 by interfering with the phosphorylation of these receptors in the plasma membrane of breast carcinoma cells. Mutational studies reveal that a positively charged C-terminal melittin sequence mediates plasma membrane interaction and anticancer activity. Engineering of an RGD motif further enhances targeting of melittin to malignant cells with minimal toxicity to normal cells. Lastly, administration of melittin enhances the effect of docetaxel in suppressing breast tumor growth in an allograft model. Our work unveils a molecular mechanism underpinning the anticancer selectivity of melittin, and outlines treatment strategies to target aggressive breast cancers.

Consequences of a short time exposure to a sublethal dose of Flupyradifurone (Sivanto) pesticide early in life on survival and immunity in the honeybee (Apis mellifera).

Dramatic losses of pollinating insects have become of global concern, as they threaten not only key ecosystem services but also human food production. Recent research provided evidence that interactions between ecological stressors are drivers of declining pollinator health and responsible for observed population collapses. We used the honeybee Apis mellifera and conducted a series of experiments to test for long-term effects of a single short exposure to the agricultural pesticide flupyradifurone to a second environmental stressor later in life. To do this, we exposed individuals during their larval development or early adulthood to sublethal dosages of flupyradifurone (0.025 µg for larvae and 0.645 µg for imagos), either pure or as part of an agricultural formulation (Sivanto). We afterwards exposed bees to a second ecological stressor infecting individuals with 10,000 spores of the fungal gut parasite Nosema ceranae. We found that pesticide exposures significantly reduced survival of bees and altered the expression of several immune and detoxification genes. The ability of bees to respond to these latter effects differed significantly between colonies, offering opportunities to breed bees with elevated levels of pesticide tolerance in the future. We conclude that short episodes of sublethal pesticide exposures during development are sufficient to trigger effects later in life and could therefore contribute to the widespread declines in bee health.

Seminal fluid compromises visual perception in honeybee queens reducing their survival during additional mating flights.

Queens of social insects make all mate-choice decisions on a single day, except in honeybees whose queens can conduct mating flights for several days even when already inseminated by a number of drones. Honeybees therefore appear to have a unique, evolutionarily derived form of sexual conflict: a queen's decision to pursue risky additional mating flights is driven by later-life fitness gains from genetically more diverse worker-offspring but reduces paternity shares of the drones she already mated with. We used artificial insemination, RNA-sequencing and electroretinography to show that seminal fluid induces a decline in queen vision by perturbing the phototransduction pathway within 24-48 hr. Follow up field trials revealed that queens receiving seminal fluid flew two days earlier than sister queens inseminated with saline, and failed more often to return. These findings are consistent with seminal fluid components manipulating queen eyesight to reduce queen promiscuity across mating flights.

Putative Drone Copulation Factors Regulating Honey Bee (Apis mellifera) Queen Reproduction and Health: A Review.

Honey bees are major pollinators of agricultural and non-agricultural landscapes. In recent years, honey bee colonies have exhibited high annual losses and commercial beekeepers frequently report poor queen quality and queen failure as the primary causes. Honey bee colonies are highly vulnerable to compromised queen fertility, as each hive is headed by one reproductive queen. Queens mate with multiple drones (male bees) during a single mating period early in life in which they obtain enough spermatozoa to fertilize their eggs for the rest of their reproductive life span. The process of mating initiates numerous behavioral, physiological, and molecular changes that shape the fertility of the queen and her influence on the colony. For example, receipt of drone semen can modulate queen ovary activation, pheromone production, and subsequent worker retinue behavior. In addition, seminal fluid is a major component of semen that is primarily derived from drone accessory glands. It also contains a complex mixture of proteins such as proteases, antioxidants, and antimicrobial proteins. Seminal fluid proteins are essential for inducing post-mating changes in other insects such as Drosophila and thus they may also impact honey bee queen fertility and health. However, the specific molecules in semen and seminal fluid that initiate post-mating changes in queens are still unidentified. Herein, we summarize the mating biology of honey bees, the changes queens undergo during and after copulation, and the role of drone semen and seminal fluid in post-mating changes in queens. We then review the effects of seminal fluid proteins in insect reproduction and potential roles for honey bee drone seminal fluid proteins in queen reproduction and health. We finish by proposing future avenues of research. Further elucidating the role of drone fertility in queen reproductive health may contribute towards reducing colony losses and advancing honey bee stock development.

Protein-Level Interactions as Mediators of Sexual Conflict in Ants.

All social insects with obligate reproductive division of labor evolved from strictly monogamous ancestors, but multiple queen-mating (polyandry) arose de novo, in several evolutionarily derived lineages. Polyandrous ant queens are inseminated soon after hatching and store sperm mixtures for a potential reproductive life of decades. However, they cannot re-mate later in life and are thus expected to control the loss of viable sperm because their lifetime reproductive success is ultimately sperm limited. In the leaf-cutting ant Atta colombica,, the survival of newly inseminated sperm is known to be compromised by seminal fluid of rival males and to be protected by secretions of the queen sperm storage organ (spermatheca). Here we investigate the main protein-level interactions that appear to mediate sperm competition dynamics and sperm preservation. We conducted an artificial insemination experiment and DIGE-based proteomics to identify proteomic changes when seminal fluid is exposed to spermathecal fluid followed by a mass spectrometry analysis of both secretions that allowed us to identify the sex-specific origins of the proteins that had changed in abundance. We found that spermathecal fluid targets only seven (2%) of the identified seminal fluid proteins for degradation, including two proteolytic serine proteases, a SERPIN inhibitor, and a semen-liquefying acid phosphatase. In vitro, and in vivo, experiments provided further confirmation that these proteins are key molecules mediating sexual conflict over sperm competition and viability preservation during sperm storage. In vitro, exposure to spermathecal fluid reduced the capacity of seminal fluid to compromise survival of rival sperm in a matter of hours and biochemical inhibition of these seminal fluid proteins largely eliminated that adverse effect. Our findings indicate that A. colombica, queens are in control of sperm competition and sperm storage, a capacity that has not been documented in other animals but is predicted to have independently evolved in other polyandrous social insects.

Synergistic effects of pathogen and pesticide exposure on honey bee (Apis mellifera) survival and immunity.

Declines in native insect pollinator populations and substantial losses in managed honey bees have been reported on a global scale and become a widespread concern because of the importance of these insects for human food production and ecosystem stability. Several potential factors have been studied as possible causes of declining pollinator health, such as parasites and pathogens, exposure to agricultural pesticides, habitat loss and/or climate change. More recently, a combination of these factors rather than a single cause have been blamed for observed pollinator losses, but field studies of such interactions are challenging, especially in the presence of confounding environmental stressors. We therefore examined the impact of single and combined stressors on the honey bee (Apis mellifera) in a generally healthy Australian population. We exposed workers during their larval development and drones until they reached sexual maturity to the neonicotinoid pesticide Thiamethoxam, at concentrations more than 20 times lower than we initially measured in the field, the microsporidian gut pathogen Nosema apis or both stressors at the same time. We found that simultaneous exposure significantly reduced bee health. We observed a substantial increase in mortality and a reduction of immunocompetence in workers exposed to both the pathogen and the pesticide. We conclude that the exposure of generally healthy bees to multiple environmental stressors results in synergistic effects where the effects are expected to negatively impact performance and could be sufficient to trigger colony collapse. We found that the vast majority of males did not survive to sexual maturity after exposure to very low levels of Thiamethoxam. This would not only reduce the reproductive success of individual colonies, but can also impact gene flow and genetic diversity at the population level, which are both known as key components of honey bee health.

Rival seminal fluid induces enhanced sperm motility in a polyandrous ant.

BACKGROUND: Promiscuous mating and sperm competition often induce arms races between the sexes with detrimental outcomes for females. However, ants with multiply-inseminated queens have only a single time-window for sperm competition and queens are predicted to gain control over the outcome of sperm storage quickly. The seminal fluid of Acromyrmex leaf-cutting ants reduces the viability of rival sperm, but how confrontations between unrelated ejaculates affect sperm storage remains unknown. RESULTS: We investigated the effects of ejaculate admixture on sperm motility in A. echinatior and found that the proportion of motile spermatozoa, sperm swimming speed, and linearity of sperm movement increased when rival ejaculates were mixed in vitro. Major effects induced by the seminal fluid of rival males were of similar magnitude to those generated by queen reproductive tract secretions, whereas own seminal fluid induced lower sperm activation levels. CONCLUSIONS: Our results suggest that ant sperm respond via a self-non-self recognition mechanism to similar or shared molecules expressed in the reproductive secretions of both sexes. Lower sperm motility in the presence of own seminal fluid indicates that enhanced motility is costly and may trade-off with sperm viability during sperm storage, consistent with studies in vertebrates. Our results imply that ant spermatozoa have evolved to adjust their energetic expenditure during insemination depending on the perceived level of sperm competition.

Differential immune gene expression in sperm storage organs of leaf-cutting ants.

Leaf-cutting ant queens mate with multiple males during a single nuptial flight and store sperm for up to two decades. During mating, males transfer sperm from their accessory testes to the queen bursa copulatrix from where it enters the spermatheca, an insect sperm storage organ that has become highly specialized in long-lived ant queens who never re-mate later in life. Long-term storage without the possibility to obtain new sperm creates an immune defence dilemma, because recognition of non-self cells eliminates infections but may also target irreplaceable sperm and reduce lifetime reproductive success. We therefore hypothesized that non-specific immune responses, like pathogen melanization, should be silenced in the spermatheca, because they rely on general non-self recognition, and that specific responses such as antimicrobial peptides are activated instead as they specifically target pathogenic bacteria and/or fungi. The maintenance of uninfected sperm cells by males before mating is not constrained by non-self recognition, meaning immune regulation might be more liberal in male reproductive organs. To test this hypothesis, we measured gene expression of two antimicrobial peptides, abaecin and defensin, and prophenoloxidase, an important enzyme of the melanization pathway, in male accessory glands and testes and in queen bursae copulatrix and spermathecae of Acromyrmex echinatior and Atta colombica leaf-cutting ants. As expected, prophenoloxidase expression was low in reproductive organs that sustain prolonged contact with sperm, whereas antimicrobial peptides showed average to high expression, indicating that leaf-cutting ants invest in specific rather than generalist immune defences for pathogen protection in organs that store sperm.

Multi-modal imaging and analysis in the search for iron-based magnetoreceptors in the honeybee Apis mellifera.

The honeybee Apis mellifera is one of many animal species for which empirical evidence of a magnetic sense has been provided. The underlying mechanisms postulated for magnetoreception in bees are varied, but most point towards the abdomen as the most likely anatomical region for its location, partly owing to the large accumulation of iron in trophocyte cells that comprise the honeybee fat body. Using a multi-modal imaging and analysis approach, we have investigated iron in the honeybee, with a particular focus on the abdomen and the utility of such techniques as applied to magnetoreception. Abdominal iron is shown to accumulate rapidly, reaching near maximum levels only 5 days after emerging from the comb and is associated with the accumulation of iron within the fat body. While fat body iron could be visualized, no regions of interest, other than perhaps the fat body itself, were identified as potential sites for magnetoreceptive cells. If an iron-based magnetoreceptor exists within the honeybee abdomen the large accumulation of iron in the fat body is likely to impede its discovery.

Insights into the molecular basis of long-term storage and survival of sperm in the honeybee (Apis mellifera).

Honeybee males produce ejaculates consisting of large numbers of high quality sperm. Because queens never re-mate after a single mating episode early in life, sperm are stored in a specialised organ for years but the proximate mechanisms underlying this key physiological adaptation are unknown. We quantified energy metabolism in honeybee sperm and show that the glycolytic metabolite glyceraldehyde-3-phosphate (GA3P) is a key substrate for honeybee sperm survival and energy production. This reliance on non-aerobic energy metabolism in stored sperm was further supported by our findings of very low levels of oxygen inside the spermatheca. Expression of GA3P dehydrogenase (GAPDH), the enzyme involved in catabolism of GA3P, was significantly higher in stored compared to ejaculated sperm. Therefore, long-term sperm storage seems facilitated by the maintenance of non-aerobic energy production, the need for only the ATP-producing steps of glycolysis and by avoiding sperm damage resulting from ROS production. We also confirm that honeybee sperm is capable of aerobic metabolism, which predominates in ejaculated sperm while they compete for access to the spermatheca, but is suppressed during storage. Consequently, the remarkable reproductive traits of honeybees are proximately achieved by differential usage of energy production pathways to maximise competitiveness and minimise damage of sperm.

Infections with the Sexually Transmitted Pathogen Nosema apis Trigger an Immune Response in the Seminal Fluid of Honey Bees (Apis mellifera).

Honey bee (Apis mellifera) males are highly susceptible to infections with the sexually transmitted fungal pathogen Nosema apis. However, they are able to suppress this parasite in the ejaculate using immune molecules in the seminal fluid. We predicted that males respond to infections by altering the seminal fluid proteome to minimize the risk to sexually transmit the parasite to the queen and her colony. We used iTRAQ isotopic labeling to compare seminal fluid proteins from infected and noninfected males and found that N. apis infections resulted in significant abundance changes in 111 of the 260 seminal fluid proteins quantitated. The largest group of proteins with significantly changed abundances consisted of 15 proteins with well-known immune-related functions, which included two significantly more abundant chitinases in the seminal fluid of infected males. Chitinases were previously hypothesized to be involved in honey bee antifungal activity against N. apis. Here we show that infection with N. apis triggers a highly specific immune response in the seminal fluid of honey bee males.

Differential proteomics reveals novel insights into Nosema-honey bee interactions.

Host manipulation is a common strategy by parasites to reduce host defense responses, enhance development, host exploitation, reproduction and, ultimately, transmission success. As these parasitic modifications can reduce host fitness, increased selection pressure may result in reciprocal adaptations of the host. Whereas the majority of studies on host manipulation have explored resistance against parasites (i.e. ability to prevent or limit an infection), data describing tolerance mechanisms (i.e. ability to limit harm of an infection) are scarce. By comparing differential protein abundance, we provide evidence of host-parasite interactions in the midgut proteomes of N. ceranae-infected and uninfected honey bees from both Nosema-tolerant and Nosema-sensitive lineages. We identified 16 proteins out of 661 protein spots that were differentially abundant between experimental groups. In general, infections of Nosema resulted in an up-regulation of the bee's energy metabolism. Additionally, we identified 8 proteins that were differentially abundant between tolerant and sensitive honey bees regardless of the Nosema infection. Those proteins were linked to metabolism, response to oxidative stress and apoptosis. In addition to bee proteins, we also identified 3 Nosema ceranae proteins. Interestingly, abundance of two of these Nosema proteins were significantly higher in infected Nosema-sensitive honeybees relative to the infected Nosema-tolerant lineage. This may provide a novel candidate for studying the molecular interplay between N. ceranae and its honey bee host in more detail.

Flight behaviour of honey bee (Apis mellifera) workers is altered by initial infections of the fungal parasite Nosema apis.

Honey bees (Apis mellifera) host a wide range of parasites, some being known contributors towards dramatic colony losses as reported over recent years. To counter parasitic threats, honey bees possess effective immune systems. Because immune responses are predicted to cause substantial physiological costs for infected individuals, they are expected to trade off with other life history traits that ultimately affect the performance and fitness of the entire colony. Here, we tested whether the initial onset of an infection negatively impacts the flight behaviour of honey bee workers, which is an energetically demanding behaviour and a key component of foraging activities. To do this, we infected workers with the widespread fungal pathogen Nosema apis, which is recognised and killed by the honey bee immune system. We compared their survival and flight behaviour with non-infected individuals from the same cohort and colony using radio frequency identification tags (RFID). We found that over a time frame of four days post infection, Nosema did not increase mortality but workers quickly altered their flight behaviour and performed more flights of shorter duration. We conclude that parasitic infections influence foraging activities, which could reduce foraging ranges of colonies and impact their ability to provide pollination services.

Queen reproductive tract secretions enhance sperm motility in ants.

Queens of Acromyrmex leaf-cutting ants store sperm of multiple males after a single mating flight, and never remate even though they may live for decades and lay tens of thousands of eggs. Sperm of different males are initially transferred to the bursa copulatrix and compete for access to the long-term storage organ of queens, but the factors determining storage success or failure have never been studied. We used in vitro experiments to show that reproductive tract secretions of Acromyrmex echinatior queens increase sperm swimming performance by at least 50% without discriminating between sperm of brothers and unrelated males. Indiscriminate female-induced sperm chemokinesis makes the likelihood of storage directly dependent on initial sperm viability and thus provides a simple mechanism to secure maximal possible reproductive success of queens, provided that initial sperm motility is an accurate predictor of viability during later egg fertilization.

Sperm use economy of honeybee (Apis mellifera) queens.

The queens of eusocial ants, bees, and wasps only mate during a very brief period early in life to acquire and store a lifetime supply of sperm. As sperm cannot be replenished, queens have to be highly economic when using stored sperm to fertilize eggs, especially in species with large and long-lived colonies. However, queen fertility has not been studied in detail, so that we have little understanding of how economic sperm use is in different species, and whether queens are able to influence their sperm use. This is surprising given that sperm use is a key factor of eusocial life, as it determines the fecundity and longevity of queens and therefore colony fitness. We quantified the number of sperm that honeybee (Apis mellifera) queens use to fertilize eggs. We examined sperm use in naturally mated queens of different ages and in queens artificially inseminated with different volumes of semen. We found that queens are remarkably efficient and only use a median of 2 sperm per egg fertilization, with decreasing sperm use in older queens. The number of sperm in storage was always a significant predictor for the number of sperm used per fertilization, indicating that queens use a constant ratio of spermathecal fluid relative to total spermathecal volume of 2.364 × 10(-6) to fertilize eggs. This allowed us to calculate a lifetime fecundity for honeybee queens of around 1,500,000 fertilized eggs. Our data provide the first empirical evidence that honeybee queens do not manipulate sperm use, and fertilization failures in worker-destined eggs are therefore honest signals that workers can use to time queen replacement, which is crucial for colony performance and fitness.

Seminal fluid of honeybees contains multiple mechanisms to combat infections of the sexually transmitted pathogen Nosema apis.

The societies of ants, bees and wasps are genetically closed systems where queens only mate during a brief mating episode prior to their eusocial life and males therefore provide queens with a lifetime supply of high-quality sperm. These ejaculates also contain a number of defence proteins that have been detected in the seminal fluid but their function and efficiency have never been investigated in great detail. Here, we used the honeybee Apis mellifera and quantified whether seminal fluid is able to combat infections of the fungal pathogen Nosema apis, a widespread honeybee parasite that is also sexually transmitted. We provide the first empirical evidence that seminal fluid has a remarkable antimicrobial activity against N. apis spores and that antimicrobial seminal fluid components kill spores in multiple ways. The protein fraction of seminal fluid induces extracellular spore germination, which disrupts the life cycle of N. apis, whereas the non-protein fraction of seminal fluid induces a direct viability loss of intact spores. We conclude that males provide their ejaculates with efficient antimicrobial molecules that are able to kill N. apis spores and thereby reduce the risk of disease transmission during mating. Our findings could be of broader significance to master honeybee diseases in managed honeybee stock in the future.

Magnetic particle-mediated magnetoreception.

Behavioural studies underpin the weight of experimental evidence for the existence of a magnetic sense in animals. In contrast, studies aimed at understanding the mechanistic basis of magnetoreception by determining the anatomical location, structure and function of sensory cells have been inconclusive. In this review, studies attempting to demonstrate the existence of a magnetoreceptor based on the principles of the magnetite hypothesis are examined. Specific attention is given to the range of techniques, and main animal model systems that have been used in the search for magnetite particulates. Anatomical location/cell rarity and composition are identified as two key obstacles that must be addressed in order to make progress in locating and characterizing a magnetite-based magnetoreceptor cell. Avenues for further study are suggested, including the need for novel experimental, correlative, multimodal and multidisciplinary approaches. The aim of this review is to inspire new efforts towards understanding the cellular basis of magnetoreception in animals, which will in turn inform a new era of behavioural research based on first principles.