A Honey Bee (Apis mellifera) Light Phase Response Curve

The authors report a phase response curve (PRC) for individual honey bees (Apis mellifera) to single 1-h light pulses (1000 lux) using an Aschoff Type 1 protocol (n?=?134). The bee PRC is a weak (Type 1) PRC with a maximum advance of 1.5?h between circadian time (CT) 18 and 3 and a maximum delay of 1.5?h between CT 12 and 18. This is the first published honey bee light PRC and provides an important resource for chronobiologists and honey bee researchers. It may also have practical applications for what is an economically important species frequently transported across different time zones. (Author correspondence: G.warman@auckland.ac.nz)     

The effects of the general anaesthetic isoflurane on the honey bee (Apis mellifera) circadian clock

General anaesthesia administered during the day has previously been shown to phase shift the honey bee clock. We describe a phase response curve for honey bees (n=105) to six hour isoflurane anaesthesia. The honey bee isoflurane PRC is “weak” with a delay portion (maximum shift of –1.88 hours, circadian time 0 – 3) but no advance zone. The isoflurane-induced shifts observed here are in direct opposition to those of light. Furthermore, concurrent administration of light and isoflurane abolishes the shifts that occur with isoflurane alone. Light may thus provide a means of reducing isoflurane–induced phase shifts.     

Probing the circadian pacemaker of a mouse using two light pulses

In two separate sets of experiments, the phases of the locomotor activity rhythm of the nocturnal field mouse Mus booduga were probed using two light pulses (LPs). In the first set of experiments, the circadian pacemaker underlying the locomotor activity rhythm was perturbed at circadian time 14 (CT 14) using a resetting light pulse LP1 of 1000 lux intensity and 15 min duration. The phases of the resetting pacemaker were then probed at all even CTs between CT 16 and CT 14 using a PRC probing light pulse LP2 of equal strength. The "LP2 PRC" thus obtained was then compared with the single light pulse PRC in terms of the area under delay (D) and advance (A) zones of the PRCs. The time course and waveform of the two LP PRCs suggest that the LP2 PRC resembled the single LP PRC, displaced by 2 h toward the right. The LP1 PRC had smaller D compared to the single LP PRC (p = 0.007), whereas both the PRCs had A of equal magnitude (p = 0.23). This suggests that the pacemaker phase shifts rapidly after LP perturbations. In the second set of experiments, the LP1 was administered at CT 14. The phase of the pacemaker was then perturbed on day 1 (next cycle after LP1) either 2 h after activity onset (at ca. CT 14 of the transient cycle) or 8 h after activity onset (at ca. CT 20 of the transient cycle) using an LP2 of equal strength. It was observed that the steady-state phase shifts evoked by positioning an LP2, 2 h after activity onset, were positively correlated with the phase shifts observed on day 1. The steady-state phase shifts observed, when the LP2 was positioned, 8 h after activity onset, were negatively correlated with the phase shifts observed on day 1. These results suggest that the transient cycles do not mirror the state of the pacemaker oscillator.     

Phase and period responses to short light pulses in a wild diurnal rodent,Funambulus pennanti

Photic phase response curves (PRCs) have been extensively studied in many laboratory-bred diurnal and nocturnal rodents. However, comparatively fewer studies have addressed the effects of photic cues on wild diurnal mammals. Hence, we studied the effects of short durations of light pulses on the circadian systems of the diurnal Indian Palm squirrel, Funambulus pennanti. Adult males entrained to a light–dark cycle (12?h–12?h) were transferred to constant darkness (DD). Free-running animals were exposed to brief light pulses (250 lux) of 15?min, 3 circadian hours (CT) apart (CT 0, 3, 6, 9, 12, 15, 18 and 21). Phase shifts evoked at different phases were plotted against CT and a PRC was constructed. F. pennanti exhibited phase-dependent phase shifts at all the CTs studied, and the PRC obtained was of type 1 at the intensity of light used. Phase advances were evoked during the early subjective day and late subjective night, while phase delays occurred during the late subjective day and early subjective night, with maximum phase delay at CT 15 (?2.04?±?0.23?h), and maximum phase advance at CT 21 (1.88?±?0.31?h). No dead zone was seen at this resolution. The free-running period of the rhythm was concurrently lengthened (deceleration) during the late subjective day and early subjective night, while period shortening (acceleration) occurred during the late subjective night. The maximum deceleration was noticed at CT 15 (?0.40?±?0.09?h) and the maximum acceleration at CT 21 (0.39?±?0.07?h). A significant positive correlation exists between the phase shifts and the period changes (r?=?0.684, p?=?0.001). The shapes of both the PRC and period response curve (τRC) qualitatively resemble each other. This suggests that the palm squirrel’s circadian system is entrained both by phase and period responses to light. Thus, F. pennanti exhibits robust clock-resetting in response to light pulses.     

Formal properties of the circadian and photoperiodic systems of Japanese quail: phase response curve and effects of T-cycles.

A role for the circadian system in photoperiodic time measurement in Japanese quail is controversial. The authors undertook studies of the circadian and photoperiodic system of Japanese quail to try to identify a role for the circadian system in photoperiodic time measurement. The circadian studies showed that the circadian system acts like a low-amplitude oscillator: It is readily reset by light without significant transients, has a Type 0 phase response curve (PRC), and has a large range of entrainment. In fact, a cycle length that is often used in resonance protocols (LD 6:30) is within the range of entrainment. The authors employed T-cycle experiments; that is, LD cycles with 6- and 14-h photoperiods and period lengths ranging from 18 to 36 h to test for circadian involvement in photoperiodic time measurement. The results did not give evidence for circadian involvement in photoperiodic time measurement: T-cycles utilizing 6-h photoperiods were uniformly noninductive (that is, did not stimulate the reproductive system), whereas T-cycles utilizing 14-h photoperiods were inductive (stimulatory). A good match was observed between the phase-angles exhibited on the T-cycles employing 6-h photoperiods and the predicted phase-angles calculated from a PRC generated from 6-h light pulses.     

Influence of honey bee (Hymenoptera: Apidae) density on the production of canola (Crucifera: Brassicacae)

Pollination is an essential step in the seed production of canola, Brassica napus L. It is achieved with the assistance of various pollen vectors, but particularly by the honey bee, Apis mellifera L. Although the importance of pollination has been shown for the production of seed crops, the need to introduce bee hives in canola fields during flowering to increase oil seed yield has not yet been proven. With the purpose of showing this, hives of A. mellifera were grouped and placed in various canola fields in the Chaudière-Appalaches and Capitale-Nationale regions (nine fields; three blocks with three treatments; 0, 1.5, and 3 hives per hectare). A cage was used to exclude pollinators and bee visitations were observed in each field. After the harvest, yield analyses were done in relation to the bee density gradient created, by using pod set, number of seeds per plant, and weight of 1000 seeds. Results showed an improvement in seed yield of 46% in the presence of three honey bee hives per hectare, compared with the absence of hives. The introduction of honey bees contributed to production and consequently, these pollinators represented a beneficial and important pollen vector for the optimal yield of canola.     

Does Cry1Ac Bt‐toxin impair development of worker larvae of Africanized honey bee?

The western honey bee (Apis mellifera L.) is a widespread pollinator species. The present study aimed to test if Africanized honey bee larvae are negatively affected by the ingestion of diet contaminated with the Bacillus thunringiensis toxin Cry1Ac, which is expressed in GM cotton plants. The toxin activity was confirmed in bioassays with the velvetbean caterpillar (Anticarsia gemmatalis), a soybean pest species susceptible to Cry1Ac. The honey bee larvae were subjected to ingestion of either pure larval diet (control), diluted larval diet (diluted control) or larval diet diluted in a Cry1Ac solution at a concentration compatible with the maximum possible field exposure. Although diluted diet slightly increased larval mortality, Cry1Ac ingestion did not affect survival, developmental time, and neither adult body mass nor size, indicating that GM plants are unlikely to significantly impair the development of honey bee larvae. The larval‐rearing system reported here was suitable to assess the lethal and sub‐lethal effects of GM expressed toxins against honey bee larvae.     

A Cell Line Resource Derived from Honey Bee (Apis mellifera) Embryonic Tissues

A major hindrance to the study of honey bee pathogens or the effects of pesticides and nutritional deficiencies is the lack of controlled in vitro culture systems comprised of honey bee cells. Such systems are important to determine the impact of these stress factors on the developmental and cell biology of honey bees. We have developed a method incorporating established insect cell culture techniques that supports sustained growth of honey bee cells in vitro. We used honey bee eggs mid to late in their embryogenesis to establish primary cultures, as these eggs contain cells that are progressively dividing. Primary cultures were initiated in modified Leibovitz’s L15 medium and incubated at 32^°C. Serial transfer of material from several primary cultures was maintained and has led to the isolation of young cell lines. A cell line (AmE-711) has been established that is composed mainly of fibroblast-type cells that form an adherent monolayer. Most cells in the line are diploid (2n = 32) and have the Apis mellifera karyotype as revealed by Giemsa stain. The partial sequence for the mitochondrial-encoded cytochrome c oxidase subunit I (Cox 1) gene in the cell line is identical to those from honey bee tissues and a consensus sequence for A. mellifera. The population doubling time is approximately 4 days. Importantly, the cell line is continuously subcultured every 10–14 days when split at a 1:3 ratio and is cryopreserved in liquid nitrogen. The cell culture system we have developed has potential application for studies aimed at honey bee development, genetics, pathogenesis, transgenesis, and toxicology.     

Do competing honey bees matter? Dynamics and abundance of native bees before and after honey bee invasion

To provide replicate samples of local bee populations in a nature preserve, light traps operated continuously on Barro Colorado Island (BCI), Panama, collected bees for 17 years, including 10 years following invasion by African Apis mellifera. Honey bees appeared in light traps as the first swarms colonized the Panama Canal area. Their numbers followed seasonal trends shown in inde-pendent studies, thus indicating bee abundance and activity in a large area. No measurable population-level impact of competition between this invading honey bee and native bees, despite many demonstrations of resource competition at flower patch and colony levels, changed annual abundances of all 15 native bee species. Native bee abundance did not decrease, nor did native bees show substantial reciprocal yearly change with honey bee abundance. One strong negative correlation of bee catches with an extremely rainy year was found. However, multiple regression using rainfall and honey bee abundance as the independent variables showed that neither was responsible for bee population change over 17 years. Nearly half the native species declined during a year that displayed peak honey bee number. That competition from honey bees on an island the size of BCI was necessarily reduced below impact levels expected on the mainland is discussed using a model of resource and consumer density, foraging range, and island size.     

Rapid Phase Resetting of a Mammalian Orcadian Rhythm by Brief Light Pulses

The phase-shift (Δψ) responses of the circadian rhythm in the field mouse Mus booduga to brief light pulses (LPs) of 15 minutes duration and 1000 lux intensity were measured in 90 experiments. In each experiment, a resetting light pulse LP₁ was administered at CT14 (CT, circadian time), and a scanning light pulse LP₂ was then variously administered in separate experiments at CT16, CT20, and CT22 in the same and in the next circadian cycle. The Δψ obtained in all these two-pulse experiments did not differ significantly from theoretical values computed on the assumption that LP₁ reset the phase response curve (PRC) rapidly. In each case, the steady-state Δψ observed after LP₁ and LP₂ differed significantly from the Δψ obtained at the same CT in determination of the single-pulse PRC (control) and also differed significantly from the values on the assumption of no Δψ in the PRC following LP₁. These results indicate that the circadian pacemaker of M. booduga, as measured by its PRC, is substantially reset within 2h after a light pulse at CT14. (Chronobiology International 14(6), 537–548, 1997)     

The effects of temperature and dose of formic acid on treatment efficacy against Varroa destructor (Acari: Varroidae), a parasite of Apis mellifera (Hymenoptera: Apidae)

In order to decrease the variability of formic acid treatments against the honey bee parasite the varroa mite, Varroa destructor, it is necessary to determine the dose-time combination that best controls mites without harming bees. The concentration × time (CT) product is a valuable tool for studying fumigants and how they might perform under various environmental conditions. This laboratory study is an assessment of the efficacy of formic acid against the varroa mite under a range of formic acid concentrations and temperatures. The objectives are 1) to determine the effect of temperature and dose of formic acid on worker honey bee and varroa mite survival, 2) to determine the CT₅₀ products for both honey bees and varroa mites and 3) to determine the best temperature and dose to optimize selectivity of formic acid treatment for control of varroa mites. Worker honey bees and varroa mites were fumigated at 0, 0.01, 0.02, 0.04, 0.08, and 0.16 mg/L at 5, 15, 25, and 35 °C for 12 d. Mite and bee mortality were assessed at regular intervals. Both mite and bee survival were affected by formic acid dose. Doses of 0.08 and 0.16 mg/L were effective at killing mites at all temperatures tested above 5 °C. There was a significant interaction between temperature, dose, and species for the CT₅₀ product. The difference between the CT₅₀ product of bees and mites was significant at only a few temperature-dose combinations. CT product values showed that at most temperatures the greatest fumigation efficiency occurred at lower doses of formic acid. However, the best fumigation efficiency and selectivity combination for treatments occurred at a dose of 0.16 mg/L when the temperature was 35 °C. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genome-wide characterization of long intergenic non-coding RNAs (lincRNAs) provides new insight into viral diseases in honey bees Apis cerana and Apis mellifera

Background

Long non-coding RNAs (lncRNAs) are a class of RNAs that do not encode proteins. Recently, lncRNAs have gained special attention for their roles in various biological process and diseases.

Results

In an attempt to identify long intergenic non-coding RNAs (lincRNAs) and their possible involvement in honey bee development and diseases, we analyzed RNA-seq datasets generated from Asian honey bee (Apis cerana) and western honey bee (Apis mellifera). We identified 2470 lincRNAs with an average length of 1011 bp from A. cerana and 1514 lincRNAs with an average length of 790 bp in A. mellifera. Comparative analysis revealed that 5 % of the total lincRNAs derived from both species are unique in each species. Our comparative digital gene expression analysis revealed a high degree of tissue-specific expression among the seven major tissues of honey bee, different from mRNA expression patterns. A total of 863 (57 %) and 464 (18 %) lincRNAs showed tissue-dependent expression in A. mellifera and A. cerana, respectively, most preferentially in ovary and fat body tissues. Importantly, we identified 11 lincRNAs that are specifically regulated upon viral infection in honey bees, and 10 of them appear to play roles during infection with various viruses.

Conclusions

This study provides the first comprehensive set of lincRNAs for honey bees and opens the door to discover lincRNAs associated with biological and hormone signaling pathways as well as various diseases of honey bee.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1868-7) contains supplementary material, which is available to authorized users.     

Resource overlap and possible competition between honey bees and wild bees in central Europe

Evidence for interspecific competition between honey bees and wild bees was studied on 15 calcareous grasslands with respect to: (1) foraging radius of honey bees, (2) overlap in resource use, and (3) possible honey bee effects on species richness and abundance of flower-visiting, ground-nesting and trap-nesting wild bees. The grasslands greatly differed in the number of honey bee colonies within a radius of 2 km and were surrounded by agricultural habitats. The number of flower-visiting honey bees on both potted mustard plants and small grassland patches declined with increasing distance from the nearest apiary and was almost zero at a distance of 1.5–2.0 km. Wild bees were observed visiting 57 plant species, whereas honey bees visited only 24 plant species. Percentage resource overlap between honey bees and wild bees was 45.5%, and Hurlbert’s index of niche overlap was 3.1. In total, 1849 wild bees from 98 species were recorded on the calcareous grasslands. Neither species richness nor abundance of wild bees were negatively correlated with the density of honey bee colonies (within a radius of 2 km) or the density of flower-visiting honey bees per site. Abundance of flower- visiting wild bees was correlated only with the percentage cover of flowering plants. In 240 trap nests, 1292 bee nests with 6066 brood cells were found. Neither the number of bee species nor the number of brood cells per grassland was significantly correlated with the density of honey bees. Significant correlations were found only between the number of brood cells and the percentage cover of shrubs. The number of nest entrances of ground-nesting bees per square metre was not correlated with the density of honey bees but was negatively correlated with the cover of vegetation. Interspecific competition by honey bees for food resources was not shown to be a significant factor determining abundance and species richness of wild bees. Received: 22 March 1999 / Accepted: 24 September 1999     

Microsatellites in the silkworm, Bombyx mori: abundance, polymorphism, and strain characterization.

We have isolated and characterized microsatellites (simple sequence repeat (SSR) loci) from the silkworm genome. The screening of a partial genomic library by the conventional hybridization method led to the isolation of 28 microsatellites harbouring clones. The abundance of (CA)n repeats in the silkworm genome was akin to those reported in the other organisms such as honey bee, pig, and human, but the (CT)n repeat motif is less common compared to bumble bee and honey bee genomes. Detailed analysis of 13 diverse silkworm strains with a representative of 15 microsatellite loci revealed a number of alleles ranging from 3 to 17 with heterozygosity values of 0.66-0.90. Along with strain-specific microsatellite markers, diapause and non-diapause strain-specific alleles were also identified. The repeat length did not show any relationship with the degree of polymorphism in the present study. The co-dominant inheritance of microsatellite markers was demonstrated in F1 offspring. A list of primer sequences that tag each locus is provided. The availability of microsatellite markers can be expected to enhance the power and resolution of genome analysis in silkworm.     

The Potential Influence of Bumble Bee Visitation on Foraging Behaviors and Assemblages of Honey Bees on Squash Flowers in Highland Agricultural Ecosystems

Bee species interactions can benefit plant pollination through synergistic effects and complementary effects, or can be of detriment to plant pollination through competition effects by reducing visitation by effective pollinators. Since specific bee interactions influence the foraging performance of bees on flowers, they also act as drivers to regulate the assemblage of flower visitors. We selected squash (Cucurbita pepo L.) and its pollinators as a model system to study the foraging response of honey bees to the occurrence of bumble bees at two types of sites surrounded by a high amount of natural habitats (≥ 58% of land cover) and a low amount of natural habitats (≤ 12% of land cover) in a highland agricultural ecosystem in China. At the individual level, we measured the elapsed time from the departure of prior pollinator(s) to the arrival of another pollinator, the selection of honey bees for flowers occupied by bumble bees, and the length of time used by honey bees to explore floral resources at the two types of sites. At the community level, we explored the effect of bumble bee visitation on the distribution patterns of honey bees on squash flowers. Conclusively, bumble bee visitation caused an increase in elapsed time before flowers were visited again by a honey bee, a behavioral avoidance by a newly-arriving honey bee to select flowers occupied by bumble bees, and a shortened length of time the honey bee takes to examine and collect floral resources. The number of overall bumble bees on squash flowers was the most important factor explaining the difference in the distribution patterns of honey bees at the community level. Furthermore, decline in the number of overall bumble bees on the squash flowers resulted in an increase in the number of overall honey bees. Therefore, our study suggests that bee interactions provide an opportunity to enhance the resilience of ecosystem pollination services against the decline in pollinator diversity.     

Visual associative learning in restrained honey bees with intact antennae

A restrained honey bee can be trained to extend its proboscis in response to the pairing of an odor with a sucrose reward, a form of olfactory associative learning referred to as the proboscis extension response (PER). Although the ability of flying honey bees to respond to visual cues is well-established, associative visual learning in restrained honey bees has been challenging to demonstrate. Those few groups that have documented vision-based PER have reported that removing the antennae prior to training is a prerequisite for learning. Here we report, for a simple visual learning task, the first successful performance by restrained honey bees with intact antennae. Honey bee foragers were trained on a differential visual association task by pairing the presentation of a blue light with a sucrose reward and leaving the presentation of a green light unrewarded. A negative correlation was found between age of foragers and their performance in the visual PER task. Using the adaptations to the traditional PER task outlined here, future studies can exploit pharmacological and physiological techniques to explore the neural circuit basis of visual learning in the honey bee.     

Circadian phototransduction: phase resetting and frequency of the circadian clock of Gonyaulax cells in red light

Constant red light (RR) influences the Gonyaulax clock in several ways: (1) Phase resetting by white or blue light pulses is stronger under background RR than in constant white light (WW); (2) frequency of the rhythm is less in RR than in WW; and (3) the amplitude of the spontaneous flashing rhythm is greater in RR than in WW. The phase response curve (PRC) to 4-hr white or blue light pulses is of high amplitude (Type 0) for cells in RR, but is of lower amplitude (Type 1) for cells in WW. In all cases, the PRC is highly asymmetrical: The magnitude of advance phase resetting is far higher than that of delay resetting. Consistent with this PRC, Gonyaulax cells in RR (free-running period greater than 24 hr) will entrain to T cycles of between 21 and 26.5 hr. The bioluminescence rhythms exhibit "masking" by blue light pulses while entrained to these T cycles. The fluence response of phase resetting to light-pulse intensity is not linear or logarithmic--rather, it is discontinuous. This feature is consistent with a limit cycle interpretation of Type 0 resetting of circadian clocks. Light pulses that cause large phase shifts also shorten the subsequent free-running period. The phase angle difference between the clock and the previous LD cycle is within 2 hr of the same phase between 16 degrees C and 25 degrees C, as determined from the light PRCs at various temperatures. Several drugs that inhibit mitochondria and/or electron transport will partially inhibit the phase shift by light.     

Genetics,Synergists, and Age Affect Insecticide Sensitivity of the Honey Bee,Apis mellifera

The number of honey bee colonies in the United States has declined to half of its peak level in the 1940s, and colonies lost over the winter have reached levels that are becoming economically unstable. While the causes of these losses are numerous and the interaction between them is very complex, the role of insecticides has garnered much attention. As a result, there is a need to better understand the risk of insecticides to bees, leading to more studies on both toxicity and exposure. While much research has been conducted on insecticides and bees, there have been very limited studies to elucidate the role that bee genotype and age has on the toxicity of these insecticides. The goal of this study was to determine if there are differences in insecticide sensitivity between honey bees of different genetic backgrounds (Carniolan, Italian, and Russian stocks) and assess if insecticide sensitivity varies with age. We found that Italian bees were the most sensitive of these stocks to insecticides, but variation was largely dependent on the class of insecticide tested. There were almost no differences in organophosphate bioassays between honey bee stocks (<1-fold), moderate differences in pyrethroid bioassays (1.5 to 3-fold), and dramatic differences in neonicotinoid bioassays (3.4 to 33.3-fold). Synergism bioassays with piperonyl butoxide, amitraz, and coumaphos showed increased phenothrin sensitivity in all stocks and also demonstrated further physiological differences between stocks. In addition, as bees aged, the sensitivity to phenothrin significantly decreased, but the sensitivity to naled significantly increased. These results demonstrate the variation arising from the genetic background and physiological transitions in honey bees as they age. This information can be used to determine risk assessment, as well as establishing baseline data for future comparisons to explain the variation in toxicity differences for honey bees reported in the literature.     

Management increases genetic diversity of honey bees via admixture

The process of domestication often brings about profound changes in levels of genetic variation in animals and plants. The honey bee, Apis mellifera, has been managed by humans for centuries for both honey and wax production and crop pollination. Human management and selective breeding are believed to have caused reductions in genetic diversity in honey bee populations, thereby contributing to the global declines threatening this ecologically and economically important insect. However, previous studies supporting this claim mostly relied on population genetic comparisons of European and African (or Africanized) honey bee races; such conclusions require reassessment given recent evidence demonstrating that the honey bee originated in Africa and colonized Europe via two independent expansions. We sampled honey bee workers from two managed populations in North America and Europe as well as several old-world progenitor populations in Africa, East and West Europe. Managed bees had highly introgressed genomes representing admixture between East and West European progenitor populations. We found that managed honey bees actually have higher levels of genetic diversity compared with their progenitors in East and West Europe, providing an unusual example whereby human management increases genetic diversity by promoting admixture. The relationship between genetic diversity and honey bee declines is tenuous given that managed bees have more genetic diversity than their progenitors and many viable domesticated animals.