Both olfactory and visual cues promote the hornet Vespa velutina to locate its honeybee prey Apis cerana

Predators use olfactory, visual and sometimes acoustic cues from the preys to assess food information. However, it is not known if the aggressive hornets (Vespa spp.) use olfactory, visual, or both types of information to find and recognize prey. In the present study, we trained hornet workers (Vespa velutina) to a feeding area. Once the hornets began consistently foraging at this feeding area, we determined whether they located prey (bees, Apis cerana) via olfactory or visual cues. We did this by testing whether hornets were attracted to a dummy bait (bee dummy bait or non-bee dummy bait) treated with extracts of honeybee cuticular hydrocarbons. We then tested whether hornets could distinguish between bee dummy bait and cotton ball dummy bait, both treated with bee odors. Hornets preferred the dummy treated with bee odors, and bee dummies (with bee images) were more attractive to the hornet than the cotton ball dummies with only bee odors. These results clearly indicate that a combination of olfactory and visual cues helps the hornet to locate its prey.     

Analysis of GABAergic and Non-GABAergic Neuron Activity in the Optic Lobes of the Forager and Re-Orienting Worker Honeybee (Apis mellifera L.)

Background

European honeybee (Apis mellifera L.) foragers have a highly developed visual system that is used for navigation. To clarify the neural basis underlying the highly sophisticated visual ability of foragers, we investigated the neural activity pattern of the optic lobes (OLs) in pollen-foragers and re-orienting bees, using the immediate early gene kakusei as a neural activity marker.

Methodology/Principal Findings

We performed double-in situ hybridization of kakusei and Amgad, the honeybee homolog of the GABA synthesizing enzyme GAD, to assess inhibitory neural activity. kakusei-related activity in GABAergic and non-GABAergic neurons was strongly upregulated in the OLs of the foragers and re-orienting bees, suggesting that both types of neurons are involved in visual information processing. GABAergic neuron activity was significantly higher than non-GABAergic neuron activity in a part of the OLs of only the forager, suggesting that unique information processing occurs in the OLs of foragers. In contrast, GABAergic neuron activity in the antennal lobe was significantly lower than that of GABAergic neurons in the OLs in the forager and re-orienting bees, suggesting that kakusei-related visual activity is dominant in the brains of these bees.

Conclusions/Significance

The present study provides the first evidence that GABAergic neurons are highly active in the OL neurons of free-moving honeybees and essential clue to reveal neural basis of the sophisticated visual ability that is equipped in the small and simple brain.     

European honeybee defense against Japanese yellow hornet using heat generation by bee‐balling behavior

The Japanese honeybee, Apis cerana japonica Radoszkowski, uses unique generation of heat by bee‐balling to defend against, overheat and kill predacious Japanese hornets. We have now observed the European honeybee, Apis mellifera Linnaeus, using similar bee‐balling behavior and heat generation against the Japanese yellow hornet, Vespa simillima xanthoptera Cameron. We monitored temperatures in the center of the bee‐ball and inside thoracic muscles of the captured hornet and found that the thoracic internal temperature (45.8 ± 2.32°C) was higher than that of the bee‐ball (44.0 ± 0.96°C). Although the thoracic temperature of captured hornets rose to the upper lethal level, defending European bees also showed some stinging attempts against the hornet, unlike the sympatric Japanese honeybee, which never stings during bee‐balling. The European honeybee bee‐balling behavior consists of three phases: (i) heating; (ii) heat‐retaining; and (iii) break up. Our results suggest that European honeybees kill hornets by raising the body temperature of hornets rapidly without stinging. The tactics of bee‐balling against hornets are complex and may be performed by extended division of labor.     

In Situ Hybridization Analysis of the Expression of Futsch,Tau, and MESK2 Homologues in the Brain of the European Honeybee (Apis mellifera L.)

Background

The importance of visual sense in Hymenopteran social behavior is suggested by the existence of a Hymenopteran insect-specific neural circuit related to visual processing and the fact that worker honeybee brain changes morphologically according to its foraging experience. To analyze molecular and neural bases that underlie the visual abilities of the honeybees, we used a cDNA microarray to search for gene(s) expressed in a neural cell-type preferential manner in a visual center of the honeybee brain, the optic lobes (OLs).

Methodology/Principal Findings

Expression analysis of candidate genes using in situ hybridization revealed two genes expressed in a neural cell-type preferential manner in the OLs. One is a homologue of Drosophila futsch, which encodes a microtubule-associated protein and is preferentially expressed in the monopolar cells in the lamina of the OLs. The gene for another microtubule-associated protein, tau, which functionally overlaps with futsch, was also preferentially expressed in the monopolar cells, strongly suggesting the functional importance of these two microtubule-associated proteins in monopolar cells. The other gene encoded a homologue of Misexpression Suppressor of Dominant-negative Kinase Suppressor of Ras 2 (MESK2), which might activate Ras/MAPK-signaling in Drosophila. MESK2 was expressed preferentially in a subclass of neurons located in the ventral region between the lamina and medulla neuropil in the OLs, suggesting that this subclass is a novel OL neuron type characterized by MESK2-expression. These three genes exhibited similar expression patterns in the worker, drone, and queen brains, suggesting that they function similarly irrespective of the honeybee sex or caste.

Conclusions

Here we identified genes that are expressed in a monopolar cell (Amfutsch and Amtau) or ventral medulla-preferential manner (AmMESK2) in insect OLs. These genes may aid in visualizing neurites of monopolar cells and ventral medulla cells, as well as in analyzing the function of these neurons.     

Comparison of learning and memory of Apis cerana and Apis mellifera

The honeybee is an excellent model organism for research on learning and memory among invertebrates. Learning and memory in honeybees has intrigued neuroscientists and entomologists in the last few decades, but attention has focused almost solely on the Western honeybee, Apis mellifera. In contrast, there have been few studies on learning and memory in the Eastern honeybee, Apis cerana. Here we report comparative behavioral data of color and grating learning and memory for A. cerana and A. mellifera in China, gathered using a Y-maze apparatus. We show for the first time that the learning and memory performance of A. cerana is significantly better on both color and grating patterns than that of A. mellifera. This study provides the first evidence of a learning and memory difference between A. cerana and A. mellifera under controlled conditions, and it is an important basis for the further study of the mechanism of learning and memory in honeybees.     

Honey Bees Modulate Their Olfactory Learning in the Presence of Hornet Predators and Alarm Component

In Southeast Asia the native honey bee species Apis cerana is often attacked by hornets (Vespa velutina), mainly in the period from April to November. During the co-evolution of these two species honey bees have developed several strategies to defend themselves such as learning the odors of hornets and releasing alarm components to inform other mates. However, so far little is known about whether and how honey bees modulate their olfactory learning in the presence of the hornet predator and alarm components of honey bee itself. In the present study, we test for associative olfactory learning of A. cerana in the presence of predator odors, the alarm pheromone component isopentyl acetate (IPA), or a floral odor (hexanal) as a control. The results show that bees can detect live hornet odors, that there is almost no association between the innately aversive hornet odor and the appetitive stimulus sucrose, and that IPA is less well associated with an appetitive stimulus when compared with a floral odor. In order to imitate natural conditions, e.g. when bees are foraging on flowers and a predator shows up, or alarm pheromone is released by a captured mate, we tested combinations of the hornet odor and floral odor, or IPA and floral odor. Both of these combinations led to reduced learning scores. This study aims to contribute to a better understanding of the prey-predator system between A. cerana and V. velutina.     

Honeybee (Apis cerana) guards do not discriminate between robbers and reproductive parasites

A hopelessly queenless honeybee colony has only one reproductive option: some workers must produce sons before the colony dies. This requires the workers to curtail egg policing (removal of worker-produced eggs), rendering the colony vulnerable to non-natal reproductive parasitism. In the Western honeybee, Apis mellifera, guarding (prevention of foreign workers from entering a colony) increases in queenless colonies, providing a defence against non-natal parasitism. However, in the closely related Eastern honeybee A. cerana, queenless colonies appear to be more tolerant of bees from other colonies. We presented guards of four A. cerana colonies with three types of workers: nestmate returning foragers, non-nestmate returning foragers and non-nestmates from a laying-worker colony. The latter are likely to have active ovaries, allowing us to test whether guard bees can detect which potential invaders are more likely to be reproductive parasites. After assessing guards’ reactions, we recaptured test bees and dissected them to determine levels of ovary activation. We found that nestmates were accepted significantly more frequently than the other two types of workers. However, there was no difference in the overall acceptance rates of non-nestmate returning foragers and bees from within laying-worker colonies. In addition, ovary-activated workers were no less likely to be accepted than those with inactive ovaries. Interestingly, colonies were more accepting of all three types of test bee after being made queenless. We conclude that, as has been previously suggested, guarding has no specific role in the prevention of non-natal parasitism in A. cerana.     

Stable nitrogen and carbon isotope ratios in wild native honeybees: the influence of land use and climate

The eastern hive bee Apis cerana is a major honeybee species in Asia providing numerous ecosystem services. Understanding how much the honeybees depend on natural and human-influenced plants and landscapes in different climates is important could contribute to evaluate how wild honeybees use food resources and to measure the ecosystem services. We investigated the effects of land use and climate changes on stable nitrogen and carbon isotope ratios in wild populations of A. cerana. In populations from 139 individual sites throughout Japan, we measured nitrogen (δ¹⁵N) and carbon (δ¹³C) stable isotope ratios and analyzed the effects of land use and climate. Our results showed that forested areas and annual precipitation had significant effects on δ¹⁵N, and that paddy fields and urban areas had significant effects on δ¹³C. These results suggest that A. cerana sensibly uses available food resources in the various environments and that stable nitrogen and carbon isotope ratios clearly reflect the effects of land use and climate changes on the populations of A. cerana. Thus, stable nitrogen and carbon isotope ratios in A. cerana, which widely distributes in Asia, can be used as indicators of the environments, such as land use and climate, of an area within its foraging range.     

The pheromones of laying workers in two honeybee sister species: <Emphasis Type="Italic">Apis cerana</Emphasis> and <Emphasis Type="Italic">Apis mellifera</Emphasis>

When a honeybee colony loses its queen, workers activate their ovaries and begin to lay eggs. This is accompanied by a shift in their pheromonal bouquet, which becomes more queen like. Workers of the Asian hive bee Apis cerana show unusually high levels of ovary activation and this can be interpreted as evidence for a recent evolutionary arms race between queens and workers over worker reproduction in this species. To further explore this, we compared the rate of pheromonal bouquet change between two honeybee sister species of Apis cerana and Apis mellifera under queenright and queenless conditions. We show that in both species, the pheromonal components HOB, 9-ODA, HVA, 9-HDA, 10-HDAA and 10-HDA have significantly higher amounts in laying workers than in non-laying workers. In the queenright colonies of A. mellifera and A. cerana, the ratios (9-ODA)/(9-ODA + 9-HDA + 10-HDAA + 10-HDA) are not significantly different between the two species, but in queenless A. cerana colonies the ratio is significant higher than in A. mellifera, suggesting that in A. cerana, the workers’ pheromonal bouquet is dominated by the queen compound, 9-ODA. The amount of 9-ODA in laying A. cerana workers increased by over 585% compared with the non-laying workers, that is 6.75 times higher than in A. mellifera where laying workers only had 86% more 9-ODA compared with non-laying workers.     

Varroa destructor changes its cuticular hydrocarbons to mimic new hosts

Varroa destructor (Vd) is a honeybee ectoparasite. Its original host is the Asian honeybee, Apis cerana, but it has also become a severe, global threat to the European honeybee, Apis mellifera. Previous studies have shown that Varroa can mimic a host''s cuticular hydrocarbons (HC), enabling the parasite to escape the hygienic behaviour of the host honeybees. By transferring mites between the two honeybee species, we further demonstrate that Vd is able to mimic the cuticular HC of a novel host species when artificially transferred to this new host. Mites originally from A. cerana are more efficient than mites from A. mellifera in mimicking HC of both A. cerana and A. mellifera. This remarkable adaptability may explain their relatively recent host-shift from A. cerana to A. mellifera.     

Honey Bee Inhibitory Signaling Is Tuned to Threat Severity and Can Act as a Colony Alarm Signal

Alarm communication is a key adaptation that helps social groups resist predation and rally defenses. In Asia, the world’s largest hornet, Vespa mandarinia, and the smaller hornet, Vespa velutina, prey upon foragers and nests of the Asian honey bee, Apis cerana. We attacked foragers and colony nest entrances with these predators and provide the first evidence, in social insects, of an alarm signal that encodes graded danger and attack context. We show that, like Apis mellifera, A. cerana possesses a vibrational “stop signal,” which can be triggered by predator attacks upon foragers and inhibits waggle dancing. Large hornet attacks were more dangerous and resulted in higher bee mortality. Per attack at the colony level, large hornets elicited more stop signals than small hornets. Unexpectedly, stop signals elicited by large hornets (SS _{large hornet}) had a significantly higher vibrational fundamental frequency than those elicited by small hornets (SS _{small hornet}) and were more effective at inhibiting waggle dancing. Stop signals resulting from attacks upon the nest entrance (SS _nest) were produced by foragers and guards and were significantly longer in pulse duration than stop signals elicited by attacks upon foragers (SS _forager). Unlike SS _forager, SS _nest were targeted at dancing and non-dancing foragers and had the common effect, tuned to hornet threat level, of inhibiting bee departures from the safe interior of the nest. Meanwhile, nest defenders were triggered by the bee alarm pheromone and live hornet presence to heat-ball the hornet. In A. cerana, sophisticated recruitment communication that encodes food location, the waggle dance, is therefore matched with an inhibitory/alarm signal that encodes information about the context of danger and its threat level.     

Imidacloprid Alters Foraging and Decreases Bee Avoidance of Predators

Concern is growing over the effects of neonicotinoid pesticides, which can impair honey bee cognition. We provide the first demonstration that sublethal concentrations of imidacloprid can harm honey bee decision-making about danger by significantly increasing the probability of a bee visiting a dangerous food source. Apis cerana is a native bee that is an important pollinator of agricultural crops and native plants in Asia. When foraging on nectar containing 40 µg/L (34 ppb) imidacloprid, honey bees (Apis cerana) showed no aversion to a feeder with a hornet predator, and 1.8 fold more bees chose the dangerous feeder as compared to control bees. Control bees exhibited significant predator avoidance. We also give the first evidence that foraging by A. cerana workers can be inhibited by sublethal concentrations of the pesticide, imidacloprid, which is widely used in Asia. Compared to bees collecting uncontaminated nectar, 23% fewer foragers returned to collect the nectar with 40 µg/L imidacloprid. Bees that did return respectively collected 46% and 63% less nectar containing 20 µg/L and 40 µg/L imidacloprid. These results suggest that the effects of neonicotinoids on honey bee decision-making and other advanced cognitive functions should be explored. Moreover, research should extend beyond the classic model, the European honey bee (A. mellifera), to other important bee species.     

Olfactory Attraction of the Hornet Vespa velutina to Honeybee Colony Odors and Pheromones

Since the beginning of the last century, the number of biological invasions has continuously increased worldwide. Due to their environmental and economical consequences, invasive species are now a major concern. Social wasps are particularly efficient invaders because of their distinctive biology and behavior. Among them, the yellow-legged hornet, Vespa velutina, is a keen hunter of domestic honeybees. Its recent introduction to Europe may induce important beekeeping, pollination, and biodiversity problems. Hornets use olfactory cues for the long-range detection of food sources, in this case the location of honeybee colonies, but the exact nature of these cues remains unknown. Here, we studied the orientation behavior of V. velutina workers towards a range of hive products and protein sources, as well as towards prominent chemical substances emitted by these food sources. In a multiple choice test performed under controlled laboratory conditions, we found that hornets are strongly attracted to the odor of some hive products, especially pollen and honey. When testing specific compounds, the honeybee aggregation pheromone, geraniol, proved highly attractive. Pheromones produced by honeybee larvae or by the queen were also of interest to hornet workers, albeit to a lesser extent. Our results indicate that V. velutina workers are selectively attracted towards olfactory cues from hives (stored food, brood, and queen), which may signal a high prey density. This study opens new perspectives for understanding hornets’ hunting behavior and paves the way for developing efficient trapping strategies against this invasive species.     

Identification and characterization of an Egr ortholog as a neural immediate early gene in the European honeybee (Apis mellifera L.)

To date, there are only few reports of immediate early genes (IEGs) available in insects. Aiming at identifying a conserved IEG in insects, we characterized an Egr homolog of the honeybee (AmEgr: Apis mellifera Egr). AmEgr was transiently induced in whole worker brains after seizure induction. In situ hybridization for AmEgr indicated that neural activity of a certain mushroom body (a higher brain center) neuron subtype, which is the same as that we previously identified using another non-coding IEG, termed kakusei, is more enhanced in forager brains. These findings suggest that Egr can be utilized as an IEG in insects.     

Infections of Nosema ceranae in four different honeybee species

The microsporidium Nosema ceranae is detected in honeybees in Thailand for the first time. This endoparasite has recently been reported to infect most Apis mellifera honeybee colonies in Europe, the US, and parts of Asia, and is suspected to have displaced the endemic endoparasite species, Nosema apis, from the western A. mellifera. We collected and identified species of microsporidia from the European honeybee (A. mellifera), the cavity nesting Asian honeybee (Apis cerana), the dwarf Asian honeybee (Apis florea) and the giant Asian honeybee (Apis dorsata) from colonies in Northern Thailand. We used multiplex PCR technique with two pairs of primers to differentiate N. ceranae from N. apis. From 80 A. mellifera samples, 62 (77.5%) were positively identified for the presence of the N. ceranae. Amongst 46 feral colonies of Asian honeybees (A. cerana, A. florea and A. dorsata) examined for Nosema infections, only N. ceranae could be detected. No N. apis was found in our samples. N. ceranae is found to be the only microsporidium infesting honeybees in Thailand. Moreover, we found the frequencies of N. ceranae infection in native bees to be less than that of A. mellifera.     

The Linker for Activation of B Cells (LAB)/Non-T Cell Activation Linker (NTAL) Regulates Triggering Receptor Expressed on Myeloid Cells (TREM)-2 Signaling and Macrophage Inflammatory Responses Independently of the Linker for Activation of T Cells

Triggering receptor expressed on myeloid cells-2 (TREM-2) is rapidly emerging as a key regulator of the innate immune response via its regulation of macrophage inflammatory responses. Here we demonstrate that proximal TREM-2 signaling parallels other DAP12-based receptor systems in its use of Syk and Src-family kinases. However, we find that the linker for activation of T cells (LAT) is severely reduced as monocytes differentiate into macrophages and that TREM-2 exclusively uses the linker for activation of B cells (LAB encoded by the gene Lat2^−/−) to mediate downstream signaling. LAB is required for TREM-2-mediated activation of Erk1/2 and dampens proximal TREM-2 signals through a novel LAT-independent mechanism resulting in macrophages with proinflammatory properties. Thus, Lat2^−/− macrophages have increased TREM-2-induced proximal phosphorylation, and lipopolysaccharide stimulation of these cells leads to increased interleukin-10 (IL-10) and decreased IL-12p40 production relative to wild type cells. Together these data identify LAB as a critical, LAT-independent regulator of TREM-2 signaling and macrophage development capable of controlling subsequent inflammatory responses.     

Genetic and phylogenetic analysis of the honey bee sacbrood virus from jiangxi isolates

The high prevalence of honeybee viral diseases poses a severe threat to the health of honeybees and causes substantial economic losses worldwide. Sacbrood virus (SBV) is a single-strand RNA virus that infects honeybees at all life stages. The infection can shorten the lifespan of adult bees and is lethal to larvae. SBV is the major cause of honeybee losses in Asia. Genetic and phylogenetic analyses of SBV isolates from different areas have been previously conducted. However, the impact of Apis mellifera Linnaeus and Apis cerana Fabricius coexistence on the infection and phylogeny of SBV remains unknown. In this study, we collected A. cerana and A. mellifera samples from commercial apiaries, only A. cerana in mountainous region. SBV prevalence was evaluated in three commercial apiaries of Jinxi, Tonggu and Nanchang and two mountainous regions of Zixi and Yifeng. In our sampling location, we found a higher SBV prevalence in the mountainous regions than in commercial apiaries. Partial structural polyprotein coding sequences were sequenced and compared with other GenBank SBV isolates. Phylogenetic tree topologies showed that SBV isolates form two major groups based on their host specificity, and isolates from same country tend to cluster together in subclades, indicating that the host and geographic region has significant effects on SBV strain specificity.     

Novel Middle-Type Kenyon Cells in the Honeybee Brain Revealed by Area-Preferential Gene Expression Analysis

The mushroom bodies (a higher center) of the honeybee (Apis mellifera L) brain were considered to comprise three types of intrinsic neurons, including large- and small-type Kenyon cells that have distinct gene expression profiles. Although previous neural activity mapping using the immediate early gene kakusei suggested that small-type Kenyon cells are mainly active in forager brains, the precise Kenyon cell types that are active in the forager brain remain to be elucidated. We searched for novel gene(s) that are expressed in an area-preferential manner in the honeybee brain. By identifying and analyzing expression of a gene that we termed mKast (middle-type Kenyon cell-preferential arrestin-related protein), we discovered novel ‘middle-type Kenyon cells’ that are sandwiched between large- and small-type Kenyon cells and have a gene expression profile almost complementary to those of large– and small-type Kenyon cells. Expression analysis of kakusei revealed that both small-type Kenyon cells and some middle-type Kenyon cells are active in the forager brains, suggesting their possible involvement in information processing during the foraging flight. mKast expression began after the differentiation of small- and large-type Kenyon cells during metamorphosis, suggesting that middle-type Kenyon cells differentiate by modifying some characteristics of large– and/or small-type Kenyon cells. Interestingly, CaMKII and mKast, marker genes for large– and middle-type Kenyon cells, respectively, were preferentially expressed in a distinct set of optic lobe (a visual center) neurons. Our findings suggested that it is not simply the Kenyon cell-preferential gene expression profiles, rather, a ‘clustering’ of neurons with similar gene expression profiles as particular Kenyon cell types that characterize the honeybee mushroom body structure.     

Attack or retreat: contrasted defensive tactics used by Cyprian honeybee colonies under attack from hornets

This study describes the tactics used by Cyprian honeybees (Apis mellifera cypria) to defend their colonies against hornet (Vespa orientalis orientalis) attacks. We use simulated hornet attacks and a combination of video recordings and image analysis to reveal, for the first time, contrasted intra-subspecies defensive tactics that operate at the colony level during predation. In some colonies, when attacked, the numbers of guards at the hive entrance increases rapidly to attack, engulf, and kill invading hornets. In other colonies, guards avoid conflicts with hornets by retreating gradually and by forming a defensive line of honeybees at the hive entrance. Retreater colonies have propolis walls at the hive entrances with small apertures that are too narrow to allow the hornet to access the hive and that therefore reinforces entrance protection. On the contrary, attacker colonies have propolis walls with large openings through which the hornet can pass; these bees block the hornet's access by intensively guarding the hive entrance. We experimentally destroy propolis walls to test whether colonies consistently rebuild walls with the same intrinsic characteristics and we also monitor the survival rate of each anti-predator tactic after massive natural predation by hornets.     

Cloning and expression pattern of odorant receptor 11 in Asian honeybee drones,Apis cerana (Hymenoptera,Apidae)

Odorant receptors play a crucial role in the special recognition of scent molecules in the honeybee olfaction system. The odorant receptor 11 (AmOR11) in western honeybee drones (Apis mellifera) has been demonstrated to specifically bind to 9-oxo-2-decenoic acid (9-ODA) of queens. However, little is known regarding the functions of OR11 Asian honeybee drones (Apis cerana) in the context of their mating activities. In this study, the odorant receptor 11 gene (AcOr11) from A. cerana was cloned, and its expression profiles were examined during two developmental stages (immature and sexually mature) and different physiological statuses (flying and crawling). The cDNA sequence of AcOr11 was highly similar to that of AmOr11, and encoded a membrane-coupled protein of 384 amino acids. The results of qRT-PCR indicated that AcOr11 was expressed at higher levels in drone antennae compared to brains, and the expression was significantly up-regulated in sexually mature drone brains compared to immature brains. Interestingly, AcOr11 expression in brains of mature flying drones was dramatically higher than those of mature crawling drones. To our knowledge, this study demonstrate a link between AcOr11 gene expression in the brain of honeybee drones and behavior associated with sexual maturity and mating flight.