Huo-Qing Zheng

Miscellaneous standard methods for Apis mellifera research

Summary A variety of methods are used in honey bee research and differ depending on the level at which the research is conducted. On an individual level, the handling of individual honey bees, including the queen, larvae and pupae are required. There are different methods for the immobilising, killing and storing as well as determining individual weight of bees. The precise timing of developmental stages is also an important aspect of sampling individuals for experiments. In order to investigate and manipulate functional processes in honey bees, e.g. memory formation and retrieval and gene expression, microinjection is often used. A method that is used by both researchers and beekeepers is the marking of queens that serves not only to help to locate her during her life, but also enables the dating of queens. Creating multiple queen colonies allows the beekeeper to maintain spare queens, increase brood production or ask questions related to reproduction. On colony level, very useful techniques are the measurement of intra hive mortality using dead bee traps, weighing of full hives, collecting pollen and nectar, and digital monitoring of brood development via location recognition. At the population level, estimation of population density is essential to evaluate the health status and using beelines help to locate wild colonies. These methods, described in this paper, are especially valuable when investigating the effects of pesticide applications, environmental pollution and diseases on colony survival.

Genome sequencing and comparative genomics of honey bee microsporidia, Nosema apis reveal novel insights into host-parasite interactions

BackgroundThe microsporidia parasite Nosema contributes to the steep global decline of honey bees that are critical pollinators of food crops. There are two species of Nosema that have been found to infect honey bees, Nosema apis and N. ceranae. Genome sequencing of N. apis and comparative genome analysis with N. ceranae, a fully sequenced microsporidia species, reveal novel insights into host-parasite interactions underlying the parasite infections.ResultsWe applied the whole-genome shotgun sequencing approach to sequence and assemble the genome of N. apis which has an estimated size of 8.5 Mbp. We predicted 2,771 protein- coding genes and predicted the function of each putative protein using the Gene Ontology. The comparative genomic analysis led to identification of 1,356 orthologs that are conserved between the two Nosema species and genes that are unique characteristics of the individual species, thereby providing a list of virulence factors and new genetic tools for studying host-parasite interactions. We also identified a highly abundant motif in the upstream promoter regions of N. apis genes. This motif is also conserved in N. ceranae and other microsporidia species and likely plays a role in gene regulation across the microsporidia.ConclusionsThe availability of the N. apis genome sequence is a significant addition to the rapidly expanding body of microsprodian genomic data which has been improving our understanding of eukaryotic genome diversity and evolution in a broad sense. The predicted virulent genes and transcriptional regulatory elements are potential targets for innovative therapeutics to break down the life cycle of the parasite.

Changes in composition of royal jelly harvested at different times: consequences for quality standards

Most of the studies on royal jelly (RJ) composition or properties as well as quality standards of commercially available royal jelly are based on RJ harvested three days (72 h) after grafting. In China, some beekeepers produce RJ harvested one (24 h) or two (48 h) days after grafting. There is a lack of knowledge about the quality of the royal jelly harvested earlier than 72 h. This study compared 32 colonies for their chemical compositions of RJ harvested at 24, 48 and 72 h after grafting, according to the proportion of moisture, protein, 10-HDA, total sugar and the value of acidity and superoxide dismutase activity. The analysis of RJ samples revealed that the composition varied significantly (for both fresh and dehydrated samples) and on some occasions above and below the range of present Chinese and Swiss standards. The results suggest that harvesting time should be considered when defining new quality standards of RJ.

Transcriptome comparison between inactivated and activated ovaries of the honey bee Apis mellifera L.

Ovarian activity not only influences fertility, but is also involved with the regulation of division of labour between reproductive and behavioural castes of female honey bees. In order to identify candidate genes associated with ovarian activity, we compared the gene expression patterns between inactivated and activated ovaries of queens and workers by means of high‐throughput RNA‐sequencing technology. A total of 1615 differentially expressed genes (DEGs) was detected between ovaries of virgin and mated queens, and more than 5300 DEGs were detected between inactivated and activated worker ovaries. Intersection analysis of DEGs amongst five libraries revealed that a similar set of genes (824) participated in the ovary activation of both queens and workers. A large number of these DEGs were predominantly related to cellular, cell and cell part, binding, biological regulation and metabolic processes. In addition, over 1000 DEGs were linked to more than 230 components of Kyoto Encyclopedia of Genes and Genomes pathways, including 25 signalling pathways. The reliability of the RNA‐sequencing results was confirmed by means of quantitative real‐time PCR. Our results provide new insights into the molecular mechanisms involved in ovary activation and reproductive division of labour.

Social apoptosis in honey bee superorganisms

Eusocial insect colonies form superorganisms, in which nestmates cooperate and use social immunity to combat parasites. However, social immunity may fail in case of emerging diseases. This is the case for the ectoparasitic mite Varroa destructor, which switched hosts from the Eastern honeybee, Apis cerana, to the Western honey bee, Apis mellifera, and currently is the greatest threat to A. mellifera apiculture globally. Here, we show that immature workers of the mite’s original host, A. cerana, are more susceptible to V. destructor infestations than those of its new host, thereby enabling more efficient social immunity and contributing to colony survival. This counterintuitive result shows that susceptible individuals can foster superorganism survival, offering empirical support to theoretical arguments about the adaptive value of worker suicide in social insects. Altruistic suicide of immature bees constitutes a social analogue of apoptosis, as it prevents the spread of infections by sacrificing parts of the whole organism, and unveils a novel form of transgenerational social immunity in honey bees. Taking into account the key role of susceptible immature bees in social immunity will improve breeding efforts to mitigate the unsustainably high colony losses of Western honey bees due to V. destructor infestations worldwide.

Extraction, partial characterization, and storage stability of β-glucosidase from propolis.

Extraction and assay conditions for β-glucosidase from propolis were optimized. Highest enzyme activity was obtained in a citric acid-disodium hydrogen phosphate buffer at pH 6.0 with 2.5% insoluble polyvinylpyrrolidone at incubation temperature of 57 °C. β-Glucosidase activities were found in all freshly harvested propolis while β-glucosidase activities were scarcely present in the randomly bought propolis. Propolis was stored at -20 °C and 4 °C for 3 mo with almost no loss of β-glucosidase activity, but at room temperature the activity decreased exponentially with the increase of storage time. These results indicated that the activity of β-glucosidase could be a candidate for propolis-freshness index. β-Glucosidase from propolis was capable of hydrolyzing p-nitrophenyl-β-D-glucoside and p-nitrophenyl-β-D-galactoside, but lacked activity toward p-nitrophenyl-β-D-glucuronide, p-nitrophenyl-β-D-cellobioside, amygdalin, cellobiose, and gentiobiose. These results were consistent with the hypothesis that flavonoid glucosides were hydrolyzed by β-glucosidase during propolis collection and processing and provided a possible explanation for why some flavonoid biosides (that is, rutin and isorhamnetin-3-O-rutinoside) exist in propolis. Practical Application: β-Glucosidase activity was detected and partial characterization of the enzyme was determined in propolis. The enzyme activity decreased exponentially with the increase of storage time at room temperature, which suggested that the activity of β-glucosidase could be regarded as a freshness index of propolis. The research will be useful for studying the chemical constituents of propolis.

Spore Loads May Not be Used Alone as a Direct Indicator of the Severity of Nosema ceranae Infection in Honey Bees Apis mellifera (Hymenoptera:Apidae).

ABSTRACT Nosema ceranae Fries et al., 1996, a microsporidian parasite recently transferred from Asian honey bees Apis cerana F., 1793, to European honey bees Apis mellifera L., 1758, has been suspected as one of the major culprits of the worldwide honey bee colony losses. Spore load is a commonly used criterion to describe the intensity of Nosema infection. In this study, by providing Nosema-infected bees with sterilized pollen, we confirmed that pollen feeding increased the spore loads of honey bees by several times either in the presence or absence of a queen. By changing the amount of pollen consumed by bees in cages, we showed that spore loads increased with an increase in pollen consumption. Nosema infections decrease honey bee longevity and transcription of vitellogenin, either with or without pollen feeding. However, the reduction of pollen consumption had a greater impact on honey bee longevity and vitellogenin level than the increase of spore counts caused by pollen feeding. These results indicate that spore loads may not be used alone as a direct indicator of the severity of N. ceranae infection in honey bees.

Evidence of Apis cerana sacbrood virus infection in Apis mellifera

ABSTRACT Sacbrood virus (SBV) is one of the most destructive viruses in the Asian honeybee Apis cerana but is much less destructive in Apis mellifera. In previous studies, SBV isolates infecting A. cerana (AcSBV) and SBV isolates infecting A. mellifera (AmSBV) were identified as different serotypes, suggesting a species barrier in SBV infection. In order to investigate this species isolation, we examined the presence of SBV infection in 318 A. mellifera colonies and 64 A. cerana colonies, and we identified the genotypes of SBV isolates. We also performed artificial infection experiments under both laboratory and field conditions. The results showed that 38 A. mellifera colonies and 37 A. cerana colonies were positive for SBV infection. Phylogenetic analysis based on RNA-dependent RNA polymerase (RdRp) gene sequences indicated that A. cerana isolates and most A. mellifera isolates formed two distinct clades but two strains isolated from A. mellifera were clustered with the A. cerana isolates. In the artificial-infection experiments, AcSBV negative-strand RNA could be detected in both adult bees and larvae of A. mellifera, although there were no obvious signs of the disease, demonstrating the replication of AcSBV in A. mellifera. Our results suggest that AcSBV is able to infect A. mellifera colonies with low prevalence (0.63% in this study) and pathogenicity. This work will help explain the different susceptibilities of A. cerana and A. mellifera to sacbrood disease and is potentially useful for guiding beekeeping practices.

A scientific note on Israeli acute paralysis virus infection of Eastern honeybee Apis cerana and vespine predator Vespa velutina

Financial support was provided to OY and PN by the COST funded project VIVA and to VD by the Qian-Tang River expert program.

Caffeic acid phenethyl ester exhibiting distinctive binding interaction with human serum albumin implies the pharmacokinetic basis of propolis bioactive components.

Caffeic acid phenethyl ester (CAPE), as one of the major bioactive components present in propolis, exhibits versatile bioactivities, especially for its potent cytotoxic effects on several cancer cell models. To understand the pharmacokinetic characteristics of CAPE, the binding interaction between CAPE and human serum albumin (HSA) was investigated in vitro using multiple spectroscopic methods and molecular docking. The results reveal that CAPE exhibits a distinctive binding interaction with HSA comparing with other propolis components. The association constant K(A) (L mol(-1)) of the binding reaches 10(6) order of magnitude, which is significantly stronger than the other components of propolis. Based on the theory of fluorescence resonance energy transfer, the binding distance was calculated as 5.7 nm, which is longer than that of the other components of propolis. The thermodynamic results indicate that the binding is mainly driven by hydrogen bonds and van der Waals force. The docking and drugs (warfarin and ibuprofen) competitive results show that CAPE is located in the subdomain IIA (Sudlows site I, FA7) of HSA, and Gln196 and Lys199 contribute to the hydrogen bonds. Circular dichroism spectra suggest an alteration of the secondary structure of HSA due to its partial unfolding in the presence of CAPE.