Honeybee colonies carrying capacity determination in north-east dry land areas of Amhara region, Ethiopia.

The objective of the study was to examine the dynamics and sugar content of nectar of major bee flora species and to determine an approximate honeybee colony carrying capacity in northeast dry land areas of the Amhara region. Acacia asak, Acacia etbaica, Acacia tortolis, Becium grandiflorum, and Cordia africana honeybee floral species were selected on the basis of relative dominance in the area. Floral nectar was collected through micropipette and washing techniques based on the flower nature of the species and nectar sugar was measured by refractometer. Hence, A.asak, A.etbaica, A.tortolis, B.grandiflorum, and C.africana could have been estimated to produce 10.2 ± 6.4 mg, 5.3 ± 4.6 mg, and 2.6±1 mg. 4, 3.7 ± 2.1 mg, and 5.7 ± 3.2mg/flower head of nectar sugar, respectively. In a single tree of A.etbaica, A.asak, A.tortolis, B.grandiflorum, and C.africana a mean of 0.15 kg, 0.15 kg, 0.06 kg, 0.01 kg, and 0.03 kg of honey yield was expected to produce respectively. Similarly in a hectare of land, a mean of 49.9 kg, 128.9 kg, 5.6 kg, 5.5 kg, and 2.2 kg of honey was estimated to harvest. In a hectare of land a sum of 57.5 kg, 57.5 kg, and 128.9 kg of honey in highland, midland, and lowland locations, respectively was estimated to produce during the main harvesting season of the area. The mean number of honeybee colonies introduced in the lowland have estimated to be 18 traditional, 6 transitional, and 5 modern hives, and in the highland 12 traditional, 6 transitional, and 5 modern hives whereas in the midland 8 traditional, 5 transitional, 3 modern hives. In summary, even in the study area with limited rainfall and high temperatures, these species secreted a substantial amount of nectar sugar and supporting the enhancement of honey yield.

A New Isolated Fungus and Its Pathogenicity for Apis mellifera Brood in China.

In this article, we report the pathogenicity of a new strain of fungus, Rhizopus oryzae to honeybee larvae, isolated from the chalkbrood-diseased mummies of honeybee larvae and pupae collected from apiaries in China. Based on morphological observation and internal transcribed spacer (ITS) region analyses, the isolated pathogenic fungus was identified as R. oryzae. Koch's postulates were performed to determine the cause-and-effect pathogenicity of this isolate fungus. The in vitro pathogenicity of this virulent fungus in honeybees was tested by artificially inoculating worker larvae in the lab. The pathogenicity of this new fungus for honeybee larvae was both conidial-concentration and exposure-time dependent; its highly infectious and virulent effect against the larvae was observed at 1 × 105 conidia/larva in vitro after 96 h of challenge. Using probit regression analysis, the LT50 value against the larvae was 26.8 h at a conidial concentration of 1 × 105 conidia/larva, and the LC50 was 6.2 × 103 conidia/larva. These results indicate that the new isolate of R. oryzae has considerable pathogenicity in honeybee larvae. Additionally, this report suggests that pathogenic phytofungi may harm their associated pollinators. We recommend further research to quantify the levels, mechanisms, and pathways of the pathogenicity of this novel isolated pathogen for honeybee larvae at the colony level.

Epidemiology, factors influencing prevalence and level of varroosis infestation (Varroa destructor) in honeybee (Apis mellifera) colonies in different agroecologies of Southwest Ethiopia.

Little information is available on the epidemiology of varroosis caused by Varroa mite, Varroa destructor infestation in Ethiopia, although it is a devastating honeybee disease that results in significant economic losses in beekeeping. Therefore, between October 2021 and October 2022, a cross-sectional study was carried out in different agroecology zones in Southwest Ethiopia to determine the prevalence and associated risk factors for varroosis, as well as the effects of this disease on honeybee colonies and honey production. A multivariate logistic regression analysis was performed to identify possible risk factors for the prevalence of V. destructor. A total of 384 adult honeybee and worker or drone brood samples were collected from honeybee colonies and examined using standard diagnostic techniques in the laboratory. The result shows that the prevalence of V. destructor was found to be 39.3% (95% CI 34.44-44.21) and 43.2% (38.27-48.18) in adult honeybees and brood, respectively. The major risk factors for the prevalence of V. destructor in the study areas included agroecology (OR = 5.2, 95% CI 1.75-14.85), type of hive (OR = 2.9, 95% CI 1.17-17.03), management system (OR = 4.3, 95% CI 1.23-14.70), and colony management (OR = 3.5, 95% CI 1.31-9.14). The lower level of colony infestation in adult bees and brood was measured as 1.97 ± 0.14 and 3.19 ± 0.25, respectively. Season, colony status, colony management, and agroecology were among the determinant factors of the level of varroa mite infestation in adult bees and brood. The results of the study demonstrated that honey production losses are largely attributable to V. destructor infestation. Therefore, it is critical to inform the community about the effects of V. destructor on honey production and develop and implement effective management strategies for this disease. In addition, further research should be done to identify and isolate additional factors that contribute to varroosis in honeybees in different regions.

StcU-2 Gene Mutation via CRISPR/Cas9 Leads to Misregulation of Spore-Cyst Formation in Ascosphaera apis.

Ascosphaera apis is the causative agent of honey bee chalkbrood disease, and spores are the only known source of infections. Interference with sporulation is therefore a promising way to manage A. apis. The versicolorin reductase gene (StcU-2) is a ketoreductase protein related to sporulation and melanin biosynthesis. To study the StcU-2 gene in ascospore production of A. apis, CRISPR/Cas9 was used, and eight hygromycin B antibiotic-resistant transformants incorporating enhanced green fluorescent protein (EGFP) were made and analyzed. PCR amplification, gel electrophoresis, and sequence analysis were used for target gene editing analysis and verification. The CRISPR/Cas9 editing successfully knocked out the StcU-2 gene in A. apis. StcU-2 mutants had shown albino and non-functional spore-cyst development and lost effective sporulation. In conclusion, editing of StcU-2 gene has shown direct relation with sporulation and melanin biosynthesis of A. apis; this effective sporulation reduction would reduce the spread and pathogenicity of A. apis to managed honey bee. To the best of our knowledge, this is the first time CRISPR/Cas9-mediated gene editing has been efficiently performed in A. apis, a fungal honey bee brood pathogen, which offers a comprehensive set of procedural references that contributes to A. apis gene function studies and consequent control of chalkbrood disease.

Significant transcriptional changes in mature daughter Varroa destructor mites during infestation of different developmental stages of honeybees.

BACKGROUND: Varroa destructor is considered a major cause of honeybee (Apis mellifera) colony losses worldwide. Although V. destructor mites exhibit preference behavior for certain honeybee lifecycle stages, the mechanism underlying host finding and preference remains largely unknown. RESULTS: By using a de novo transcriptome assembly strategy, we sequenced the mature daughter V. destructor mite transcriptome during infestation of different stages of honeybees (brood cells, newly emerged bees and adult bees). A total of 132 779 unigenes were obtained with an average length of 2745 bp and N50 of 5706 bp. About 63.1% of the transcriptome could be annotated based on sequence homology to the predatory mite Metaseiulus occidentalis proteins. Expression analysis revealed that mature daughter mites had distinct transcriptome profiles after infestation of different honeybee stages, and that the majority of the differentially expressed genes (DEGs) of mite infesting adult honeybees were down-regulated compared to that infesting the sealed brood cells. Gene ontology and KEGG pathway enrichment analyses showed that a large number of DEGs were involved in cellular process and metabolic process, suggesting that Varroa mites undergo metabolic adjustment to accommodate the cellular, molecular and/or immune response of the honeybees. Interestingly, in adult honeybees, some mite DEGs involved in neurotransmitter biosynthesis and transport were identified and their levels of expression were validated by quantitative polymerase chain reaction (qPCR). CONCLUSION: These results provide evidence for transcriptional reprogramming in mature daughter Varroa mites during infestation of honeybees, which may be relevant to understanding the mechanism underpinning adaptation and preference behavior of these mites for honeybees. © 2020 Society of Chemical Industry.

Transcriptome profiling reveals insertional mutagenesis suppressed the expression of candidate pathogenicity genes in honeybee fungal pathogen, Ascosphaera apis.

Chalkbrood disease is caused by Ascosphaera apis which severely affects honeybee brood. Spore inoculation experiments shown pathogenicity varies among different strains and mutants, however, the molecular mechanism of pathogenicity is unclear. We sequenced, assembled and annotated the transcriptomes of wild type (SPE1) and three mutants (SPE2, SPE3 and SPE4) with reduced pathogenicity that were constructed in our previous study. Illumina sequencing generated a total of 394,910,604 clean reads and de novo Trinity-based assembled into 12,989 unigenes, among these, 9,598 genes were successfully annotated to known proteins in UniProt database. A total of 172, 3,996, and 650 genes were up-regulated and 4,403, 2,845, and 3,016 genes were down-regulated between SPE2-SPE1, SPE3-SPE1, and SPE4-SPE1, respectively. Overall, several genes with a potential role in fungal pathogenicity were detected down-regulated in mutants including 100 hydrolytic enzymes, 117 transcriptional factors, and 47 cell wall related genes. KEGG pathway enrichment analysis reveals 216 genes involved in nine pathways were down-regulated in mutants compared to wild type. The down-regulation of more pathways involved in pathogenicity in SPE2 and SPE4 than SPE3 supports their lower pathogenicity during in-vitro bioassay experiment. Expression of 12 down-regulated genes in mutants was validated by quantitative real time PCR. This study provides valuable information on transcriptome variation caused by mutation for further functional validation of candidate pathogenicity genes in A. apis.

Purification of Chinese Sacbrood Virus (CSBV), Gene Cloning and Prokaryotic Expression of its Structural Protein VP1.

The aim of this study was to purify the Chinese Sacbrood Virus Beijing Miyun (BJMY-CSBV) from infected Apis cerana larvae, clone structural protein gene VP1 (named BJMY-CSBV-VP1), and investigate its biological information. The result indicated that the capsid of CSBV is of spherical shape. Gene clone experiment showed that the BJMY-CSBV-VP1 gene sequence comprised 945 bp, encoding 315 amino acids with relative molecular weight of 35.59 kDa and isoelectric point 9.38 pI. Phylogenetic analysis of amino acid sequences showed that the BJMY-CSBV-VP1 and LNDD_2015 were grouped together. Protein secondary structure prediction showed that the gene contained two α-helices, thirteen ß-folds, six polypeptide binding sites, and no disulfide bridge. Simultaneously, the BJMY-CSBV-VP1 was ligated to the expression vector pET32a(+) and then transformed into the Escherichia coli BL21 (DE3) for prokaryotic expression. The optimal expression experiment revealed that the protein was found in the inclusion body. The recombinant protein was successfully purified by washing buffer combined with supersonic fragmentation. In this study, we obtained the purified BJMY-CSBV particles, cloned BJMY-CSBV-VP1 gene, investigated the detailed information of the gene by analyzing the sequence, and obtained the purified recombinant protein, which could help for further understanding of the function of the structural protein gene VP1.

Comparative study on the dynamics and performances of Apis mellifera jemenitica and imported hybrid honeybee colonies in southwestern Saudi Arabia.

The aims of this study were to assess the seasonal population dynamics and evaluate the performance of Apis mellifera jemenitica (local bee) and introduced hybrid honeybee colonies in the lowlands and highlands of southwestern Saudi Arabia. Data regarding the performance and population dynamics parameters such as brood and adult bee population, amounts of stored pollen and nectar were gathered from the two races (25 colonies of each) for one year (April 2013 through March 2014), and statistically tested. The results indicated that at low lands, local bee colonies maintained relatively high brood and adult bee populations (P < 0.05) than introduced honeybee colonies and produced more (P < 0.05) honey. The local bee colonies were able to hoard three times more (P < 0.05) pollen and built more (P < 0.05) queen cells than introduced bees in both the low and highland areas. The annual survival rate of local bee colonies was almost double (P < 0.05) than that of introduced honeybee colonies. Moreover, local bees had greater (P < 0.05) adult bee and brood populations than imported, throughout the year. The relatively good performance of local colonies could be due to their long year's adaptation to cope with resource scarcity and unpredictable environmental conditions of the regions. The possible reasons for the dwindling of the imported hybrid colonies could be due to continuing to exhibit adaptive characteristics of their original that might not fit well with the new environment.

Nectar secretion dynamics and honey production potentials of some major honey plants in Saudi Arabia.

The contribution of a bee plant species to honey production depends on the plant's nectar secretion quality and quantity, which is mainly governed by biotic and abiotic factors. The aim of the current study, was to investigate the nectar secretion dynamics and honey production potential of 14 major bee plant species of the target area. We examined the quantity and dynamics of nectar sugar per flower five times a day using a nectar sugar washing technique and direct measuring of nectar with calibrated capillary tubes. The average nectar sugar amount of the species varied from 0.41 mg/flower to 7.7 mg/flower (P < 0.0001). The honey sugar per flower was used to extrapolate the honey production potential per plant and per hectare of land. Accordingly the honey production potential of the species observed to vary from 14 kg/hectare in Otostegia fruticosa to 829 kg/hectare in Ziziphus spina-christi. The nectar secretion dynamics of the species generally showed an increasing trend early in the morning, peaking toward midday, followed by a decline but different species observed to have different peak nectar secretion times. Generally, the tree species secreted more nectar sugar/flower than the herbs. The nectar secretion amount of the species was positively correlated with the ambient temperature, indicating the adaptation of the species to hot climatic conditions. However, different species were observed to have a different optimum temperature for peak nectar secretion. Despite the limited rainfall and high temperature of the area, many plants were found to have good potential for honey production. The monetary value of honey per hectare of the studied honeybee plant species can be of equal or greater than the per-hectare monetary value of some cultivated crops that require numerous inputs. In addition, the information generated is believed to be useful in apiary site selection and to estimate the honey bee colony carrying capacity of an area.

Brain metabolomic profiling of eastern honey bee (Apis cerana) infested with the mite Varroa destructor.

The mite Varroa destructor is currently the greatest threat to apiculture as it is causing a global decrease in honey bee colonies. However, it rarely causes serious damage to its native hosts, the eastern honey bees Apis cerana. To better understand the mechanism of resistance of A. cerana against the V. destructor mite, we profiled the metabolic changes that occur in the honey bee brain during V. destructor infestation. Brain samples were collected from infested and control honey bees and then measured using an untargeted liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based global metabolomics method, in which 7918 and 7462 ions in ESI+ and ESI- mode, respectively, were successfully identified. Multivariate statistical analyses were applied, and 64 dysregulated metabolites, including fatty acids, amino acids, carboxylic acid, and phospholipids, amongst others, were identified. Pathway analysis further revealed that linoleic acid metabolism; propanoate metabolism; and glycine, serine, and threonine metabolism were acutely perturbed. The data obtained in this study offer insight into the defense mechanisms of A. cerana against V. destructor mites and provide a better method for understanding the synergistic effects of parasitism on honey bee colonies.

New approach for determination of an optimum honeybee colony's carrying capacity based on productivity and nectar secretion potential of bee forage species.

The present study was carried out to determine an optimum honeybee colony's carrying capacity of selected valleys dominated by Ziziphus spina-christi and Acacia tortilis in the Al-Baha region, Kingdom of Saudi Arabia. The study was conducted based on the assessment of the number of colonies kept, their productivities and the existing productive bee forage resources in the target valleys with its economic implication. In the existing beekeeping practice, the average number of managed honeybee colonies introduced per square kilometer was 530 and 317 during the flowering period of Z. spina-christi and A. tortilis, respectively. Furthermore, the overall ratios of productive bee forage plants to the number of honeybee colonies introduced were 0.55 and 11.12 to Ziziphus trees and A. tortilis shrubs respectively. In the existing situation the average honey production potential of 5.21 and 0.34 kg was recorded per Ziziphus and A. tortilis plants per flowering season, respectively. The present study, revealed that the number of honeybee colonies introduced in relation to the existing bee forage potential was extremely overcrowding which is beyond the carrying capacity of bee forage resources in selected valleys and it has been observed to affect the productivities and subsequent profitability of beekeeping. The study infers that, by keeping the optimum honeybee colony's carrying capacity of valleys (88 traditional hives/km(2) or 54 Langstroth hives/km(2) in Ziziphus field and 72 traditional hives/km(2) or 44 Langstroth hives/km(2) in A. tortilis field), profitability of beekeeping can be boosted up to 130.39% and 207.98% during Z. spina-christi and A. tortilis, flowering seasons, respectively.