Native Honey Bee Species Research Articles

Honey bee (Apis cerana) queen rearing methods influence queen morphological characteristics and reproductive related gene expression

The quality of the queen is crucial for the survival and growth of honey bee colonies. There have been studies related to raising queens with excellent performance since the invention of artificial queen rearing technologies. However, relatively few studies have been undertaken that evaluate the relationship between queen quality and queen morphology and the expression level of reproduction-related genes using different-aged brood in artificial queen rearing practices of Eastern honey bees (Apis cerana). Therefore, we transferred eggs, 1-day-old larvae, 2-day-old larvae, and 3-day-old larvae to queen cells to rear queens until the queen emerged. We measured 12 indicators (the acceptance rate, birth weight, length, caliber and maximum diameter of queen cells, thorax length, width and weight, head width, wing length and width, and number of ovarian tubes). Moreover, the relative expression levels of Hexamerin, Vitellogenin, and Transferrin were measured using fat bodies and ovaries dissected from the newly emerged virgin queens. The results showed that queens reared from eggs exhibited the best morphological indexes and occupied the most abundant reproductive related gene expression level. Parameter values for queen from 1-day-old larvae were significantly higher than those for queens from 2-day-old larvae and 3-day-old larvae, and there was no significant difference between values for queens from 2-day-old larvae and 3-day-old larvae in most data, except for the relative gene expression. In conclusion, as the age of the brood increased, the quality of the queen bees declined. Rearing queens from fertilized eggs are far more likely to yield a better outcome for queen performance and colony function. This study provides data support for raising high-quality queens of Apis cerana, which would be benefit for the protection and better utilization of our native honey bee species.

The presence of identical deformed wing virus sequence variants in co-occurring Apis species in Northern Thailand may represent a potential epidemiological threat to native honey bees of Southeast Asia

Widespread native honey bee species in South and East Asia (Apis cerana, Apis dorsata and Apis florea) and the imported western honey bee (Apis mellifera) share habitats and potentially also share pathogens. Chief among the threats facing A. mellifera in Europe and North America is deformed wing virus (DWV), including its two principal genotypes: A and B (DWV-A and DWV-B respectively). Though DWV-A has been recorded in Asia’s native Apis species, it is not known if DWV-B, or both DWV-A and DWV-B, are currently widespread in Asia and, if so, whether viral transmission is primarily intraspecific or interspecific. This study aims to fill these knowledge gaps by (i) determining the DWV genotype in four co-occurring Apis host species using qPCR and (ii) inferring viral transmission between them using nucleotide sequences of DWV from Apis host species collected at three independent localities in Northern Thailand. We found DWV-A and -B in all four Apis species, the exotic A. mellifera and the native A. cerana, A. dorsata and A. florea. That DWV-A sequences were identical across Apis species at the same locality, with a similar pattern for DWV-B sequences, suggests that DWV’s epidemiology is largely driven by ongoing interspecific transmission (spillover) of DWV across co-occurring native and exotic Apis species. Both genotypes of DWV represent a serious threat to Asia’s exotic and native honey bee species.

A sublethal dose of neonicotinoid imidacloprid precisely sensed and detoxified by a C-minus odorant-binding protein 17 highly expressed in the legs of Apis cerana

As a native honeybee species in East Asia, Apis cerana is essential for the stability of local agricultural and plant ecosystems by its' olfactory system for searching nectar and pollen sources. Odorant-binding proteins (OBPs) existing in the insect's olfactory system can recognize environmental semiochemicals. It was known that sublethal doses of neonicotinoid insecticides could still cause a variety of physiological and behavioral abnormalities in bees. However, the molecular mechanism of A. cerana sensing and response for insecticide has not been further investigated. In this study, we found an A. cerana OBP17 gene significantly up-regulated expressed after exposure to sublethal doses of imidacloprid based on the transcriptomics results. The spatiotemporal expression profiles showed that OBP17 was highly expressed in the legs. Competitive fluorescence binding assays showed that OBP17 had the special and high binding affinity to imidacloprid among the 24 candidate semiochemicals, and the KA value of OBP17 binding with imidacloprid reached the maximum (6.94 × 104 L/mol) at low-temperature. Thermodynamic analysis showed that the quenching mechanism changed from dynamic to static binding interaction with the increasing temperature. Meanwhile, the force changed from hydrogen bond and van der Waals force to hydrophobic interaction and electrostatic force, indicating the interaction exhibits variability and flexibility. Molecular docking showed that Phe107 contributed the most energy. RNA interference (RNAi) results showed that OBP17 knockdown significantly enhanced the electrophysiological response of the bees' forelegs to imidacloprid. Our study indicated that OBP17 could precisely touch and sense sublethal doses of neonicotinoid imidacloprid in the natural environment through its high expression in legs, and the upregulation expression of OBP17 exposure to imidacloprid probably implied that it participate in the detoxification processes of A. cerana. Also, our research enriches the theoretical knowledge of the sensing and detoxifying activities of non-target insects' olfactory sensory system to environmental sublethal doses of systemic insecticides.

Relationship between the Abundance of Native Honey Bee Species (<i>Apis cerana</i>) and Distance to Natural Forests in Hyuganatsu (<i>Citrus tamurana</i>) Orchards in Aya Town, Japan

This study aimed to examine the relationship between the ecosystem services of pollination offered by native honey bees (Apis cerana) and the distance to natural forests (as an indicator of landscape structure) in hyuganatsu (Citrus tamurana) orchards in Aya Town, Miyazaki Prefecture, Japan. Two statistical models were developed to predict the number of visits of native honey bees to hyuganatsu tree. The area-distaince model (AD-model) considered the area of natural forests and distance from natural forests and the area model (A-model) considered the total area of the surrounding natural forests, and the results of the two models were compared. The estimated parameters of both models suggested a positive effect of natural forests on pollination services. Further, the estimated effect of the landscape structure of the AD-model was greater than that of the A-model. These results suggested that the distance to natural forests is an important landscape factor for evaluating pollination services by native honey bees.

Impact of acute oral exposure to thiamethoxam on the homing, flight, learning acquisition and short-term retention of Apis cerana.

Thiamethoxam (TMX) represents the second generation of neonicotinoids that has been widely applied in agricultural activities, while how TMX alters the behavior of Apis cerana, an important native honey bee species in China, is not clear. We carried out three independent experiments to study the impact of acute oral treatment of 20 μL TMX at concentrations of 2.4 ppb (0.048 ng/bee) and 10ppb (0.2ng/bee) on the homing, flight, learning acquisition and short-term retention ability of A. cerana. The homing ability was assessed by the catch-and-release method, the flight ability was assessed by flight mills, and the learning acquisition and short-term retention were evaluated by the proboscis extension response method. When treated with 10ppb of TMX, bees had a significantly higher average homing time, mean flight velocity, flying distance, and flying duration than the control, whereas 2.4ppb concentration did not cause any significant effect on homing or flight ability. Bees treated with either 2.4ppb or 10ppb TMX had significantly lower learning acquisition and short-term retention ability. Results suggest that acute oral exposure to 10ppb of TMX altered the short-distance homing time, flight ability, and learning acquisition and short-term retention ability. Our study also highlights the concern that acute oral exposure to a low concentration of 2.4ppb could have consequences on the behavior of A. creana. Those multiple sublethal alterations on A. cerana's behavior indicate that TMX are likely having complex but negative consequences on bee health in the field. © 2019 Society of Chemical Industry.

Yearlong association of Apis dorsata and Apis florea with flowering plants: planted forest vs. agricultural landscape

The yearlong association of two native honey bee species (Apis dorsata and A. florea) with 49 plant species was recorded in a planted forest and adjacent agricultural landscape at Multan, Pakistan. The study resulted in 588 interactions of A. dorsata with 40 plant species and 454 interactions of A. florea on 38 plant species. The most visited plants species by A. dorsata included Helianthus annuus, Citrus reticulata, Trifolium alexandrinum, Moringa oleifera and Calotropis procera, while the most visited plant species by A. florea included C. procera, Mangifera indica, T. alexandrinum, Coriandrum sativum and H. annuus. The peak abundance of bees and floral resources (i.e. number of plant species in flowering and abundance of floral units) was recorded during early March to late May followed by a gradual decline until December. Monthly abundance of both bee species was positively related to the floral resources, negatively related to relative humidity while it was not significantly related to temperature. The current study may serve as a baseline to track the degradation in ecosystem service of cross pollination and making new conservation strategies at local scale while future research should focus on tempo-spatial variations in foraging preferences, floral constancy and effect of foraging competition on crop pollination in different ecological regions of Pakistan.

Differential gene expressions of innate immune related genes of the Asian honeybee, Apis cerana, latently infected with sacbrood virus

The Asian honeybee, Apis cerana, is a native honeybee species in Korea which is important in agriculture for pollination and honey production. Since 2009, A. cerana in Korea has been endangered because of the outbreak of sacbrood virus (SBV) disease which caused serious colony loss. SBV belongs to the genus Iflavirus, and infects the honeybee larvae specifically to cause larval death. For better understanding of the molecular biological aspects of the viral pathogen and insect host, the A. cerana larvae latently infected with SBV virus (AcSBV) and control (AcCtrl) were subjected to high-throughput transcriptome sequencing to compare the gene expression profiles. 101 base paired-end Illumina sequencing generated a total of 211,850,996 and 231,925,174 clean reads from AcSBV and AcCtrl, corresponding to 17.4 Gb and 18.7 Gb of nucleotides, respectively. An in silico cDNA library was constructed by Trinity de novo assembler and TGICL clustering program to make a reference cDNA library containing 67,750 contigs. The quality-filtered Illumina short reads were mapped to the cDNA library, and the top 20,000 of the most frequently mapped contigs by the Bowtie2 program were subjected to the ImmunoDB database to annotate insect innate immune-related genes. The genes with expression levels which were two fold higher or lower in AcSBV compared to the AcCtrl, overall a total of 20 contigs were identified and annotated to demonstrate that the latent infection of SBV affects expression pattern of innate immune-related genes.

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.

A neonicotinoid impairs olfactory learning in Asian honey bees (Apis cerana) exposed as larvae or as adults.

Xenobiotics such as the neonicotinoid pesticide, imidacloprid, are used globally, but their effects on native bee species are poorly understood. We studied the effects of sublethal doses of imidacloprid on olfactory learning in the native honey bee species, Apis cerana, an important pollinator of agricultural and native plants throughout Asia. We provide the first evidence that imidacloprid can impair learning in A. cerana workers exposed as adults or as larvae. Adults that ingested a single imidacloprid dose as low as 0.1 ng/bee had significantly reduced olfactory learning acquisition, which was 1.6-fold higher in control bees. Longer-term learning (1-17 h after the last learning trial) was also impaired. Bees exposed as larvae to a total dose of 0.24 ng/bee did not have reduced survival to adulthood. However, these larval-treated bees had significantly impaired olfactory learning when tested as adults: control bees exhibited up to 4.8-fold better short-term learning acquisition, though longer-term learning was not affected. Thus, sublethal cognitive deficits elicited by neonicotinoids on a broad range of native bee species deserve further study.

Biological Mitigation of Sacbrood Disease on Apis cerana Colonies

Apis cerana is native honey bee species in Korea. This honey bee species has well adapted to cold winter. Apis cerana honey is preferred by domestic consumers. However, this honey bee species has been declined by sacbrood disease. Sacbrood is caused by a virus. Until now there has not been any chemical to treat this disease except biological method and the selection of sacbrood resistant bee stocks. This research was conducted from June to August, 2014 at the A. cerana apiary of NAAS, Suwon, Republic of Korea. Sacbrood disease on A. cerana colonies was observed and biological method was applied to treat the disease. The results of our study showed that the rate of infected colonies was from 26.0% to 87.0%. By applying requeen measure, the rate of recovered colonies was 57.1%, while the method of caged queen got only 28.6%.

Pesticide-induced oxidative stress in laboratory and field populations of native honey bees along intensive agricultural landscapes in two Eastern Indian states

Pesticides have been cited as one of the major drivers of pollinator loss. However, little is known about pesticide impacts on natural populations of native honey bee species. This study looked into the effect of pesticides with respect to oxidative stress in the laboratory and in field populations of two native Indian honey bee species (Apis dorsata and A. cerana) by examining a combination of biomarkers, e.g., superoxide dismutase, catalase and xanthine oxidase. A significant upregulation of all three biomarkers was observed in both treated individuals in laboratory experiments and field populations sampled from a pesticide use gradient. This study reports, for the first time, an increase in expression of xanthine oxidase in an invertebrate system (honey bees) exposed to pesticides.

Comparative Transcriptome Analysis of Queen, Worker, and Larva of Asian Honeybee, Apis cerana

The Asian honeybee, Apis cerana, is a native honeybee species in Korea which is important in agriculture for pollination and honey production. For better understanding of the physiology of A. cerana, high-throughput Illumina transcriptome sequencing was performed to analyze the gene expression profiles of queen, worker, and larva. A total of 219,799,682 clean reads corresponding to 22.2 Gb of nucleotide sequences was obtained from the whole body total RNA samples. The Apis mellifera reference mRNA sequence database was used to measure the gene expression level with Bowtie2 and eXpress software, and the Illumina short reads were then mapped to 11,459 out of 11,736 A. mellifera reference genes. Total of 9,221 genes with FPKM value greater than 5 of each sample group were subjected to eggNOG with BLASTX for gene ontology analysis. The differential gene expression between queen and worker, and worker and larva were analyzed to screen the overexpressed genes in each sample group. In the queen and worker sample group, total of 1,766 genes were differentially expressed with 887 and 879 genes overexpressed over two folds in queen and worker, respectively. In the worker and larva sample group, total of 1,410 genes were differentially expressed with 1,009 and 401 genes overexpressed over two folds in worker and larva, respectively.

Three species of native Thai honey bees exploit overlapping pollen resources: identification of bee flora from pollen loads and midguts from Apis cerana, A. dorsata and A. florea

SummaryIn this study, we investigated the bee flora utilised by the Thai honey bees, Apis cerana, A. dorsata and A. florea in Nan province, northern Thailand, through the identification of pollen grains from their pollen loads and midguts. We compared the pollen grains morphologically to match with local flowering plants and determined their protein concentration using a Bradford assay. The results showed that 8 families and 15 species were found by pollen load analysis whilst 12 families and 25 species were found from pollen grains of the bee midguts. The greatest number of bee flora was found from pollen loads of A. cerana, (11 species), while there were fewer for A. florea (10 species) and the fewest with A. dorsata (6 species). The highest number of bee flora species identified from pollen was found in the midgut of A. cerana, with 19 species, while A. florea had 13 and A. dorsata with only 11. The results show that the major pollen source plants of the three native honey bee species of Thailand were Mimosa pudica L., M. pigra, Celosia argentea L., Zea mays L., Wedelia trilobata L. and Syzygium malaccense L. The protein content ranged from 31.85 ± 0.83 to 48.44 ± 0.81 mg/100 mg pollen. The most abundant pollen source was from M. pudica L., perhaps because of the flower structure, shape, size, long blooming season, wide distribution, high protein concentration (43.31 ± 0.79 mg/100 mg pollen) or a combination of these characteristics.

The Prevalence of Parasites and Pathogens in Asian Honeybees Apis cerana in China

Pathogens and parasites represent significant threats to the health and well-being of honeybee species that are key pollinators of agricultural crops and flowers worldwide. We conducted a nationwide survey to determine the occurrence and prevalence of pathogens and parasites in Asian honeybees, Apis cerana, in China. Our study provides evidence of infections of A. cerana by pathogenic Deformed wing virus (DWV), Black queen cell virus (BQCV), Nosema ceranae, and C. bombi species that have been linked to population declines of European honeybees, A. mellifera, and bumble bees. However, the prevalence of DWV, a virus that causes widespread infection in A. mellifera, was low, arguably a result of the greater ability of A. cerana to resist the ectoprasitic mite Varroa destructor, an efficient vector of DWV. Analyses of microbial communities from the A. cerana digestive tract showed that Nosema infection could have detrimental effects on the gut microbiota. Workers infected by N. ceranae tended to have lower bacterial quantities, with these differences being significant for the Bifidobacterium and Pasteurellaceae bacteria groups. The results of this nationwide screen show that parasites and pathogens that have caused serious problems in European honeybees can be found in native honeybee species kept in Asia. Environmental changes due to new agricultural practices and globalization may facilitate the spread of pathogens into new geographic areas. The foraging behavior of pollinators that are in close geographic proximity likely have played an important role in spreading of parasites and pathogens over to new hosts. Phylogenetic analyses provide insights into the movement and population structure of these parasites, suggesting a bidirectional flow of parasites among pollinators. The presence of these parasites and pathogens may have considerable implications for an observed population decline of Asian honeybees.