Juvenile hormone pathway in honey bee larvae: A source of possible signal molecules for the reproductive behavior of Varroa destructor

The parasitic mite Varroa destructor devastates honey bee (Apis mellifera) colonies around the world. Entering a brood cell shortly before capping, the Varroa mother feeds on the honey bee larvae. The hormones 20‐hydroxyecdysone (20E) and juvenile hormone (JH), acquired from the host, have been considered to play a key role in initiating Varroa''s reproductive cycle. This study focuses on differential expression of the genes involved in the biosynthesis of JH and ecdysone at six time points during the first 30 hr after cell capping in both drone and worker larvae of A. mellifera. This time frame, covering the conclusion of the honey bee brood cell invasion and the start of Varroa''s ovogenesis, is critical to the successful initiation of a reproductive cycle. Our findings support a later activation of the ecdysteroid cascade in honey bee drones compared to worker larvae, which could account for the increased egg production of Varroa in A. mellifera drone cells. The JH pathway was generally downregulated confirming its activity is antagonistic to the ecdysteroid pathway during the larva development. Nevertheless, the genes involved in JH synthesis revealed an increased expression in drones. The upregulation of jhamt gene involved in methyl farnesoate (MF) synthesis came into attention since the MF is not only a precursor of JH but it is also an insect pheromone in its own right as well as JH‐like hormone in Acari. This could indicate a possible kairomone effect of MF for attracting the mites into the drone brood cells, along with its potential involvement in ovogenesis after the cell capping, stimulating Varroa''s initiation of egg laying.     

Identification of Candidate Agents Active against N. ceranae Infection in Honey Bees: Establishment of a Medium Throughput Screening Assay Based on N. ceranae Infected Cultured Cells

Many flowering plants in both natural ecosytems and agriculture are dependent on insect pollination for fruit set and seed production. Managed honey bees (Apis mellifera) and wild bees are key pollinators providing this indispensable eco- and agrosystem service. Like all other organisms, bees are attacked by numerous pathogens and parasites. Nosema apis is a honey bee pathogenic microsporidium which is widely distributed in honey bee populations without causing much harm. Its congener Nosema ceranae was originally described as pathogen of the Eastern honey bee (Apis cerana) but jumped host from A. cerana to A. mellifera about 20 years ago and spilled over from A. mellifera to Bombus spp. quite recently. N. ceranae is now considered a deadly emerging parasite of both Western honey bees and bumblebees. Hence, novel and sustainable treatment strategies against N. ceranae are urgently needed to protect honey and wild bees. We here present the development of an in vitro medium throughput screening assay for the identification of candidate agents active against N. ceranae infections. This novel assay is based on our recently developed cell culture model for N. ceranae and coupled with an RT-PCR-ELISA protocol for quantification of N. ceranae in infected cells. The assay has been adapted to the 96-well microplate format to allow automated analysis. Several substances with known (fumagillin) or presumed (surfactin) or no (paromomycin) activity against N. ceranae were tested as well as substances for which no data concerning N. ceranae inhibition existed. While fumagillin and two nitroimidazoles (metronidazole, tinidazole) totally inhibited N. ceranae proliferation, all other test substances were inactive. In summary, the assay proved suitable for substance screening and demonstrated the activity of two synthetic antibiotics against N. ceranae.     

Insight into probiotic properties of lactic acid bacterial endosymbionts of Apis mellifera L. derived from the Polish apiary

Taking into account that fructophilic lactic acid bacteria (FLAB) can play an important role in the health of honey bees and can be used as probiotics, phenotypic properties of probiotic interest of Lactobacillus kunkeei (12 strains) and Fructobacillus fructossus bacteria (2 strains), isolated from Apis mellifera gastrointestinal tract, have been studied. We have evaluated survival of tested FLAB in honey bee gut, their susceptibility to antibiotics (ampicillin, erythromycin, tylosin), cell surface hydrophobicity, auto-aggregation ability, co-aggregation with model pathogenic bacteria, biofilm formation capacity, and effect of studied FLAB, added to sucrose syrup bee diet, on longevity of honey bees. The tested FLAB exhibited good gastrointestinal tract tolerance and high antibiotic susceptibility, which are important criteria in the screening of probiotic candidates. It was also found that all FLAB studied have high cell surface hydrophobicity and fulfil next selection criterion for their use as probiotics. Symbionts of A. mellifera showed also auto- and co-aggregation capacities regarded as valuable features for biofilm formation and inhibition of pathogens adhesion to the bee gut cells. Biofilm-development ability is a desired characteristic of probiotic lactic acid bacteria. As indicated by quantitative crystal violet-stained microplate assay and confocal laser scanning microscopy imaging, all studied A. mellifera gut isolates exhibit a biofilm positive phenotype. Moreover, it was also documented, on honey bees kept in cages, that supplementation of A. mellifera sucrose diet with FLAB decreases mortality and improves significantly longevity of honey bees. Presented research showed that A. mellifera FLAB symbionts are good candidates for application as probiotics.     

A new gene, SRP16, differentially expressed in the spermathecae of honeybee queens (Apis mellifera) related with reproduction status

Honey bee queens have the ability to store sperm in spermathecae for fertilizing eggs throughout their life. To investigate mechanisms for sperm storage in Apis mellifera, we employed suppression subtractive hybridization (SSH) to find differentially expressed fragments in spermathecae between virgin queens and newly mated queens. A new gene, named SRP16, was obtained by joining the SSH products with 5′-RACE and 3′-RACE. SRP16 is predicted to encode a 41?kDa protein with 363 amino acid residues. Its expression was found in the spermathecae dominantly in honey bee queens but not in honey bee workers, with the highest expression found in spermathecae of virgin and newly mated queens. SRP16 expression was weak in other tissues of queens other than in the spermathecae and showed no obvious change with reproductive status of queens. The results suggest that SRP16 may play important roles in sperm storage and honey bee reproduction.     

Thinking beyond Western commercial honeybee hives: towards improved conservation of honey bee diversity

A decline of wild pollinators, along with a decline of bee diversity, has been a cause of concern among academics and governmental organizations. According to IPBES, a lack of wild pollinator data contributes to difficulties in comprehensively analyzing the regional status of wild pollinators in Africa, Latin America, Asia and Oceania. It may have also contributed to the prevailing lack of awareness of the diversity of honey bees, of which the managed Apis mellifera is often considered as “the (only) honey bee,” despite the fact that there are eight other honey bee species extant in Asia. A survey of 100 journal articles published in 2016 shows that 57% of the studies still identified A. mellifera as “the honey bee.” In total, 80% of studies were conducted solely on A. mellifera. This focus on A. mellifera has also caused the honey standard of Codex Alimentarius and the European Union to be based solely on A. mellifera, causing improper evaluation of honeys from other species. We recommend adapting current standards to reflect the diversity of honey bees and in the process correct failures in the honey market and pave the way towards improved protection of honey bee species and their habitats.     

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.     

Microbes from apiarian sources: Bacillus spp. in frass of the greater wax moth

Frass from the greater wax moth, Galleria mellonella, obtained from feral colonies of honey bees, Apis mellifera; from managed honey bee colonies; and from a laboratory culture of the wax moth was sampled for aerobic Gram-positive spore-forming rods. One hundred eighty-five strains belonging to the genus Bacillus were isolated, and most were identified. One hundred and three of the isolates were from frass from the wax moth culture, 61 were from frass from managed honey bee colonies, and 21 were from frass from feral honey bee colonies. The species most frequently isolated varied with the source. Fifty-eight isolates from frass from managed honey bee colonies were B. cereus which was isolated from this source only, but B. sphaericus was the most frequent isolate from frass from the wax moth culture. Bacillus megaterium and organisms belonging to the B. alvei-B. thiaminolyticus spectrum were the most frequent isolates from frass from feral honey bee colonies. Most strains isolated produced caprylate esterase-lipase, leucine aminopeptidase, and phosphoamidase. The numbers of isolates, the species, and the enzymatic activity of the strains varied with the source of the frass. In fact, the complete microbial complement varied with the source. These results are discussed in relation to possible roles of Bacillus spp. in the nutrition of the wax moth as well as the microbial ecology of the honey bee colony.     

Interactions between Cooccurring Lactic Acid Bacteria in Honey Bee Hives

In contrast to the honey bee gut, which is colonized by a few characteristic bacterial clades, the hive of the honey bee is home to a diverse array of microbes, including many lactic acid bacteria (LAB). In this study, we used culture, combined with sequencing, to sample the LAB communities found across hive environments. Specifically, we sought to use network analysis to identify microbial hubs sharing nearly identical operational taxonomic units, evidence which may indicate cooccurrence of bacteria between environments. In the process, we identified interactions between noncore bacterial members (Fructobacillus and Lactobacillaceae) and honey bee-specific “core” members. Both Fructobacillus and Lactobacillaceae colonize brood cells, bee bread, and nectar and may serve the role of pioneering species, establishing an environment conducive to the inoculation by honey bee core bacteria. Coculture assays showed that these noncore bacterial members promote the growth of honey bee-specific bacterial species. Specifically, Fructobacillus by-products in spent medium supported the growth of the Firm-5 honey bee-specific clade in vitro. Metabolic characterization of Fructobacillus using carbohydrate utilization assays revealed that this strain is capable of utilizing the simple sugars fructose and glucose, as well as the complex plant carbohydrate lignin. We tested Fructobacillus for antibiotic sensitivity and found that this bacterium, which may be important for establishment of the microbiome, is sensitive to the commonly used antibiotic tetracycline. Our results point to the possible significance of “noncore” and environmental microbial community members in the modulation of honey bee microbiome dynamics and suggest that tetracycline use by beekeepers should be limited.     

Drone and Worker Brood Microclimates Are Regulated Differentially in Honey Bees,Apis mellifera

Background

Honey bee (Apis mellifera) drones and workers show differences in morphology, physiology, and behavior. Because the functions of drones are more related to colony reproduction, and those of workers relate to both survival and reproduction, we hypothesize that the microclimate for worker brood is more precisely regulated than that of drone brood.

Methodology/Principal Findings

We assessed temperature and relative humidity (RH) inside honey bee colonies for both drone and worker brood throughout the three-stage development period, using digital HOBO^® Data Loggers. The major findings of this study are that 1) both drone and worker castes show the highest temperature for eggs, followed by larvae and then pupae; 2) temperature in drones are maintained at higher precision (smaller variance) in drone eggs and larvae, but at a lower precision in pupae than the corresponding stages of workers; 3) RH regulation showed higher variance in drone than workers across all brood stages; and 4) RH regulation seems largely due to regulation by workers, as the contribution from empty honey combs are much smaller compared to that from adult workers.

Conclusions/Significance

We conclude that honey bee colonies maintain both temperature and humidity actively; that the microclimate for sealed drone brood is less precisely regulated than worker brood; and that combs with honey contribute very little to the increase of RH in honey bee colonies. These findings increase our understanding of microclimate regulation in honey bees and may have implications for beekeeping practices.     

Detection of honey bee (Apis mellifera) viruses with an oligonucleotide microarray

In recent years, declines in honey bee (Apis mellifera L.) colonies have been observed to varying degrees worldwide with the worst losses in the USA being termed Colony Collapse Disorder (CCD). Pathogen load and the prevalence of honey bee viruses have been implicated in these losses and many diseased hives have multiple viruses present. We have designed and tested an oligonucleotide microarray which enables the simultaneous detection of nine honey bee viruses: Acute bee paralysis virus, Black queen cell virus, Chronic bee paralysis virus, Deformed wing virus, Kashmir bee virus, Sacbrood virus, Israel acute paralysis virus, Varroa destructor virus 1 and Slow paralysis virus. The microarray can be used to robustly diagnose nine viruses in one test.     

A revision of subspecies structure of western honey bee Apis mellifera

The taxonomy of honey bee A. mellifera contains a lot of issues due to the specificity of population structure, features of biology and resolutions of honey bee subspecies discrimination methods. There are a lot of transition zones between ranges of subspecies which led to the gradual changes of characteristics among neighbor subspecies. The modern taxonomic pattern of honey bee Apis mellifera is given in this paper. Thirty-three distinct honey bee subspecies are distributed across all Africa (11 subspecies), Western Asia and the Middle East (9 subspecies), and Europe (13 subspecies). All honey bee subspecies are subdivided into 5 evolutionary lineages: lineage A (10 subspecies) and its sublineage Z (3 subspecies), lineage M (3 subspecies), lineage C (10 subspecies), lineage O (3 subspecies), lineage Y (1 subspecies), lineage C or O (3 subspecies).     

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.     

Development of single nucleotide polymorphism markers specific to Apis mellifera (Hymenoptera: Apidae) line displaying high hygienic behavior against Varroa destructor,an ectoparasitic mite

To control Varroa destructor, an ectoparasitic mite, a honey bee line possessing high hygienic behavior (HHB) against this mite has been bred in South Korea. However, a method that can diagnose and assess the HHB line from control (the low hygienic behavior, LHB) line has not been reported yet. Thus, the objective of this study was to develop single nucleotide polymorphism (SNP) markers through whole-genome sequencing of worker bees from HHB line of A. mellifera caucasica and LHB line of A. m. carnica (Hymenoptera: Apidae). A total of 319,445,977 sequence reads were mapped to the known A. mellifera reference genome (average coverage of 87.46%). In 2,316,128 and 3,266,756 SNPs from HHB and LHB line, respectively, 20 SNPs that showed homozygosity in each line were selected and eight SNPs were used to diagnose the HHB line either by typical PCR-restriction fragment length polymorphism or allele-specific PCR. Six of remaining SNPs were of different sizes, enabling relatively easy differentiation of these two honey bee lines on typical agarose gel. Another remaining six SNPs had different sequences, including SNP sites. These SNP markers can be used to diagnose and assess V. destructor-specific HHB line of honey bees.     

Several workers lay eggs in the same brood cell in queenless honey bee (Apis mellifera) colonies

Queenless honey bee (Apis mellifera) colonies are often characterized by the presence of multiple eggs in brood cells. This is surprising because only one egg can be reared to maturity per cell. Moreover, worker honey bees cannot produce many eggs per day. There are several reasons that could explain the presence of multiple eggs in single cells: a) workers cannot control how many eggs they release; in this case we would expect all eggs to be from the same mother; b) excess eggs could be provided as food for the first larva to hatch in the absence of adequate brood care and this would again result in all eggs in one cell sharing the same mother; c) the number of cells available for oviposition may be limiting, obliging workers to lay eggs in cells that already contain eggs, resulting in eggs of mixed maternity. Here we show that the majority of brood cells in queenless colonies contain eggs from multiple mothers. Therefore our results suggest that the presence of multiple eggs in brood cells arises from a limitation on the number of suitable cells available for oviposition. Received 29 September 2008; revised 21 November 2008; accepted 24 November 2008.     

Medium for development of bee cell cultures (<Emphasis Type="Italic">Apis mellifera</Emphasis>: Hymenoptera: Apidae)

A media for the production of cell cultures from hymenopteran species such as honey bee, Apis mellifera L. (Hymenoptera: Apidae) was developed. Multiple bee cell cultures were produced when using bee larvae and pupae as starting material and modified Hert–Hunter 70 media. Cell culture systems for bees solves an impasse that has hindered efforts to isolate and screen pathogens which may be influencing or causing colony collapse disorder of bees. Multiple life stages of maturing larvae to early pupae were used to successfully establish cell cultures from the tissues of the head, thorax, and abdomen. Multiple cell types were observed which included free-floating suspensions, fibroblast-like, and epithelia-like monolayers. The final culture medium, WH2, was originally developed for hemipterans, Asian citrus psyllid, Diaphorina citri, and leafhopper, Homalodisca vitripennis cell cultures but has been shown to work for a diverse range of insect species such as bees. Bee cell cultures had various doubling times at 21–23°C ranging from 9–15 d. Deformed wing virus was detected in the primary explanted tissues, which tested negative by rt-PCR for Israeli acute paralysis virus (IAPV), Kashmir bee virus, acute bee paralysis virus, and black queen cell virus. Culture inoculation with IAPV from an isolate from Florida field samples, was detectable in cell cultures after two subcultures. Cell culture from hymenoptera species, such as bees, greatly advances the approaches available to the field of study on colony collapse disorders.     

Structural and functional differences in the antennal olfactory system of worker honey bees of Apis mellifera and Apis cerana

Olfactory cues are important sensory modalities on individual discrimination, perception, and efficient orientation to food sources in most insects. In honey bees, which are well known as eusocial insects, olfactory cues are mainly used to maintain a colony. Although much research has been reported on olfactory systems in honey bee olfaction, little is known about the differences between two major honey bee species, the European honey bee Apis mellifera and the Asian honey bee Apis cerana. In order to understand the differences of olfactory characteristics in the two species, we compared the distribution of sensory hairs on the antennae and antennal olfactory responses, using electron microscopy, electrophysiological recording and molecular expression level of odorant receptors. Our present study demonstrated that the antennae of A. cerana have more olfactory sensilla than A. mellifera, responding more strongly to various floral volatile compounds. At the molecular level, olfactory co-receptor (Orco), which makes heterodimers with other conventional olfactory receptors, is more abundantly expressed in the antenna of A. cerana than of A. mellifera. These findings extend our understanding of the olfactory systems and behavioral responses to various ecological and biological signals in two closely related honey bee species.     

Bidirectional Transfer of RNAi between Honey Bee and Varroa destructor: Varroa Gene Silencing Reduces Varroa Population

The mite Varroa destructor is an obligatory ectoparasite of the honey bee (Apis mellifera) and is one of the major threats to apiculture worldwide. We previously reported that honey bees fed on double-stranded RNA (dsRNA) with a sequence homologous to that of the Israeli acute paralysis virus are protected from the viral disease. Here we show that dsRNA ingested by bees is transferred to the Varroa mite and from mite on to a parasitized bee. This cross-species, reciprocal exchange of dsRNA between bee and Varroa engendered targeted gene silencing in the latter, and resulted in an over 60% decrease in the mite population. Thus, transfer of gene-silencing-triggering molecules between this invertebrate host and its ectoparasite could lead to a conceptually novel approach to Varroa control.     

Tetrahedral Gray Code for Visualization of Genome Information

We propose a tetrahedral Gray code that facilitates visualization of genome information on the surfaces of a tetrahedron, where the relative abundance of each -mer in the genomic sequence is represented by a color of the corresponding cell of a triangular lattice. For biological significance, the code is designed such that the -mers corresponding to any adjacent pair of cells differ from each other by only one nucleotide. We present a simple procedure to draw such a pattern on the development surfaces of a tetrahedron. The thus constructed tetrahedral Gray code can demonstrate evolutionary conservation and variation of the genome information of many organisms at a glance. We also apply the tetrahedral Gray code to the honey bee (Apis mellifera) genome to analyze its methylation structure. The results indicate that the honey bee genome exhibits CpG overrepresentation in spite of its methylation ability and that two conserved motifs, CTCGAG and CGCGCG, in the unmethylated regions are responsible for the overrepresentation of CpG.     

Infestation of Japanese Native Honey Bees by Tracheal Mite and Virus from Non-native European Honey Bees in Japan

Invasion of alien species has been shown to cause detrimental effects on habitats of native species. Insect pollinators represent such examples; the introduction of commercial bumble bee species for crop pollination has resulted in competition for an ecological niche with native species, genetic disturbance caused by mating with native species, and pathogen spillover to native species. The European honey bee, Apis mellifera, was first introduced into Japan for apiculture in 1877, and queen bees have been imported from several countries for many years. However, its effects on Japanese native honey bee, Apis cerana japonica, have never been addressed. We thus conducted the survey of honey bee viruses and Acarapis mites using both A. mellifera and A. c. japonica colonies to examine their infestation in native and non-native honey bee species in Japan. Honey bee viruses, Deformed wing virus (DWV), Black queen cell virus (BQCV), Israeli acute paralysis virus (IAPV), and Sacbrood virus (SBV), were found in both A. mellifera and A. c. japonica colonies; however, the infection frequency of viruses in A. c. japonica was lower than that in A. mellifera colonies. Based on the phylogenies of DWV, BQCV, and SBV isolates from A. mellifera and A. c. japonica, DWV and BQCV may infect both honey bee species; meanwhile, SBV has a clear species barrier. For the first time in Japan, tracheal mite (Acarapis woodi) was specifically found in the dead honey bees from collapsing A. c. japonica colonies. This paper thus provides further evidence that tracheal-mite-infested honey bee colonies can die during cool winters with no other disease present. These results demonstrate the infestation of native honey bees by parasite and pathogens of non-native honey bees that are traded globally.