Ascosphaera Apis Research Articles

Fungal microbiota isolated from native stingless bee species inhibited pathogens of Apis mellifera

Social bees can establish interactions with microorganisms to keep their colonies free of pathogens and parasites by developing different protection strategies. We explored the fungal microbiota isolated from three species of stingless bees, Tetragonisca fiebrigi, Plebeias sp., and Scaptotrigona jujuyensis, and its potential ability to suppress pathogenic microorganisms of A. mellifera, namely Paenibacillus larvae, Ascosphaera apis and Aspergillus flavus, which were tested and evaluated. Six filamentous fungal strains, Trametes hirsuta, Alternaria alternata, Curvularia spicifera, Skeletocutis sp., Alternaria tenuissima, Monascus spp., as well as the yeast Wickerhamomyces anomalus, were selected for trials and isolated from the heads of foraging bees. The fungal strains were identified by macroscopic and microscopic taxonomic characteristics and by sequencing of the ITS1–5.8S–ITS2 region of ribosomal DNA. All fungal strains inhibited these pathogens of A. mellifera. We also evaluated the effect of the secondary metabolites extracted with and without ethanol. Both metabolites showed antimicrobial properties, and our results suggest that fungi isolated from stingless bees produce bioactive compounds with antibacterial and antifungal effects that could be used to treat Apis mellifera colony diseases and maintain colony health.

Delivery mechanism can enhance probiotic activity against honey bee pathogens

Managed honey bee (Apis mellifera) populations play a crucial role in supporting pollination of food crops but are facing unsustainable colony losses, largely due to rampant disease spread within agricultural environments. While mounting evidence suggests that select lactobacilli strains (some being natural symbionts of honey bees) can protect against multiple infections, there has been limited validation at the field-level and few methods exist for applying viable microorganisms to the hive. Here, we compare how two different delivery systems—standard pollen patty infusion and a novel spray-based formulation—affect supplementation of a three-strain lactobacilli consortium (LX3). Hives in a pathogen-dense region of California are supplemented for 4 weeks and then monitored over a 20-week period for health outcomes. Results show both delivery methods facilitate viable uptake of LX3 in adult bees, although the strains do not colonize long-term. Despite this, LX3 treatments induce transcriptional immune responses leading to sustained decreases in many opportunistic bacterial and fungal pathogens, as well as selective enrichment of core symbionts including Bombilactobacillus, Bifidobacterium, Lactobacillus, and Bartonella spp. These changes are ultimately associated with greater brood production and colony growth relative to vehicle controls, and with no apparent trade-offs in ectoparasitic Varroa mite burdens. Furthermore, spray-LX3 exerts potent activities against Ascosphaera apis (a deadly brood pathogen) likely stemming from in-hive dispersal differences, whereas patty-LX3 promotes synergistic brood development via unique nutritional benefits. These findings provide a foundational basis for spray-based probiotic application in apiculture and collectively highlight the importance of considering delivery method in disease management strategies.

Peppermint oil promotes the induction of vitellogenin and defensin-1 in Apis mellifera nurse bees upon ingestion of heat-killed pathogens

Transgenerational immune priming (TGIP) in honeybees (Apis mellifera) has been attracting increasing attention as an efficient method to protect colonies from infectious diseases. This study investigated whether feeding nurse bees peppermint oil (PO) along with a cocktail of heat-killed Ascosphaera apis and Paenibacillus larvae (A + P + PO cocktail) can promote the induction of marker genes for TGIP. In the A + P + PO cocktail-fed nurse bees, CCHamide-2 was significantly upregulated in the brain, and the body weight was highly increased. Consequently, vitellogenin (Vg) and defensin-1 showed a significant upregulation in the fat body of nurse bees with the A + P + PO cocktail. Thus, our findings indicate that PO promoted the induction of Vg and defensin-1 in nurse bees upon ingestion of the A + P + PO cocktail.

The Ascosphaera apis Infection (Chalkbrood Disease) Alters the Gut Bacteriome Composition of the Honeybee.

The declining honeybee populations are a significant risk to the productivity and security of agriculture worldwide. Although there are many causes of these declines, parasites are a significant one. Disease glitches in honeybees have been identified in recent years and increasing attention has been paid to addressing the issue. Between 30% and 40% of all managed honeybee colonies in the USA have perished annually over the past few years. American foulbrood (AFB) and European foulbrood (EFB) have been reported as bacterial diseases, Nosema as a protozoan disease, and Chalkbrood and Stonebrood as fungal diseases. The study aims to compare the bacterial community related to the Nosema ceranae and Ascosphaera apis infection on the gut of the honeybee and compare it with the weakly active honeybees. The Nosema-infected honeybees contain the phyla Proteobacteria as the significantly dominant bacterial phyla, similar to the weakly active honeybees. In contrast, the Ascosphaera (Chalkbrood) infected honeybee contains large amounts of Firmicutes rather than Proteobacteria.

Ingestion of heat-killed pathogens confers transgenerational immunity to the pathogens via the vitellogenin–hypopharyngeal gland axis in honeybees

Honeybee diseases are a serious threat to beekeeping and pollination. Transgenerational immune priming (TGIP) has been attracting increasing attention as a promising strategy to protect honeybee colonies from infections. This study investigated whether feeding honeybees (Apis mellifera) with a heat-killed pathogen cocktail can provide them with transgenerational immunity to these pathogens. We found that vitellogenin (Vg) and defensin-1 were highly upregulated in nurse bees upon feeding them with a cocktail of heat-killed Ascosphaera apis and Paenibacillus larvae (A + P cocktail). Pathogen-pattern–recognition receptor genes in the Toll signaling pathway were upregulated in nurse bees upon ingestion of the A + P cocktail. In the nurse bees of the hives supplied with the A + P cocktail, Vg was upregulated in the fat body, and the defensin-1 expression and Vg uptake in the hypopharyngeal glands were induced. Consequently, the major proteins in royal jelly were upregulated. In addition, defensin-1 was upregulated in the queen larvae and young worker larvae in these hives. In correlation, the young worker larvae showed high pathogen resistance to P. larvae infection. Thus, our findings imply that introduction of a heat-killed pathogen cocktail into hives is an efficient strategy for conferring honeybees with social immunity through TGIP.

Effect of selected botanical compounds on Ascosphaera apis and Apis mellifera

Ascosphaera apis is a worldwide spread fungal pathogen that invades the honey bee brood and causes chalkbrood disease. In many countries, its control with fungicides is banned due to residue contamination of bee products and health risk. Since then, there is no alternative treatment. In this study, an antifungal activity of 10 essential oils or their components has been investigated. The agar dilution method showed considerable differences between its antifungal activity with the best results exhibited by thyme oil (MIC50 = 57.7 µL/L) followed by cedarwood oil (MIC50 = 64.9 µL/L), thyme (MIC50 = 82.5 µL/L), clove bud oil (MIC50 = 114.2 µL/L), and cinnamon oil (MIC50 = 136.6 µL/L). These botanical compounds have also been tested using a honey bee tolerance by complete exposure method. The oil tolerated the most by honey bees was from cedarwood (LC50 = 33.4 µL). The composition and quantity of main components of the oils mentioned above were determined by GC-MS/MS. Thymol is responsible for the antifungal activity of thyme oil, whereas, in clove bud and cinnamon oil, the main component is eugenol. Cedarwood oils contain primarily gama and alpha cendrene. The results suggest the analyzed compounds can be used to control multiple honey bee pathogens simultaneously due to their broad spectrum of antimicrobial activity and enhanced efficacy by synergistic effect.

Comparative analysis of 3 pollen sterilization methods for feeding bumble bees.

Pollen is an essential component of bee diets, and rearing bumble bees (Bombus spp.) for commercial use necessitates feeding pollen in mass quantities. This pollen is collected from honey bee (Apis mellifera L.) colonies because neither an artificial diet nor an economical, large-scale pollen collection process from flowers is available. The provenance of honey bee-collected pollen is often unknown, and in some cases has crossed international borders. Both deformed wing virus (DWV) and the fungal pathogen Ascosphaera apis (Claussen) Olive & Spiltoir (cause of chalkbrood disease); occur in honey bee-collected pollen, and infections have been observed in bumble bees. We used these pathogens as general surrogates for viruses and spore-forming fungal diseases to test the efficacy of 3 sterilization methods, and assessed whether treatment altered pollen quality for the bumble bee. Using honey bee-collected pollen spiked with known doses of DWV and A. apis, we compared gamma irradiation (GI), ozone fumigation (OZ), and ethylene oxide fumigation (EO) against an untreated positive control and a negative control. Following sterilization treatments, we tested A. apis spore viability, detected viral presence with PCR, and tested palatability to the bumble bee Bombus impatiens Cresson. We also measured bacterial growth from pollens treated with EO and GI. GI and EO outperformed OZ treatment in pathogen suppression. EO had the highest sterilizing properties under commercial conditions and retained palatability and supported bee development better than other treatments. These results suggest that EO sterilization reduces pathogen risks while retaining pollen quality as a food source for rearing bumble bees.

Preliminary Survey of Pathogens in the Asian Honey Bee (Apis cerana) in Thailand

Widespread parasites, along with emerging threats, globalization, and climate change, have greatly affected honey bees' health, leading to colony losses worldwide. In this study, we investigated the detection of biotic stressors (i.e., viruses, microsporidian, bacteria, and fungi) in Apis cerana by surveying the colonies across different regions of Thailand (Chiang Mai in the north, Nong Khai and Khon Kaen in the northeast, and Chumphon and Surat Thani in the south, in addition to the Samui and Pha-ngan islands). In this study, we detected ABPV, BQCV, LSV, and Nosema ceranae in A. cerana samples through RT-PCR. ABPV was only detected from the samples of Chiang Mai, whereas we found BQCV only in those from Chumphon. LSV was detected only in the samples from the Samui and Pha-ngan islands, where historically no managed bees are known. Nosema ceranae was found in all of the regions except for Nong Khai and Khon Kaen in northeastern Thailand. Paenibacillus larvae and Ascosphaera apis were not detected in any of the A. cerana samples in this survey. The phylogenetic tree analysis of the pathogens provided insights into the pathogens' movements and their distribution ranges across different landscapes, indicating the flow of pathogens among the honey bees. Here, we describe the presence of emerging pathogens in the Asian honey bee as a valuable step in our understanding of these pathogens in terms of the decline in eastern honey bee populations.

Prevalence of honey bee pathogens and parasites in South Korea: A five-year surveillance study from 2017 to 2021

Honey bees play an important role in the pollination of crops and wild plants and provide important products to humans. Pathogens and parasites are the main factors that threaten beekeeping in South Korea. Therefore, a nationwide detection of 14 honey bee pathogens, including parasites (phorid flies, Nosema ceranae, and Acarapis woodi mites), viruses, bacteria, and fungal pathogens, was conducted from 2017 to 2021 in the country. The infection rate and the trend of detection of each pathogenic agent were determined. A total of 830 honey bee samples from Apis cerana (n = 357) and A. mellifera (n = 473) were examined. N. ceranae (35.53%), deformed wing virus (52.63%), sacbrood virus (SBV) (52.63%), and black queen cell virus (55.26%) were the most prevalent honey bee pathogens, and their prevalence rapidly increased from 2017 to 2021. The prevalence of Paenibacillus larvae, Israeli acute paralysis virus, Ascosphaera apis, A. woodi, Melissococcus plutonius, and chronic bee paralysis virus remained stable during the surveillance period, with infection rates ranging from 5.26% to 16.45% in 2021. Other pathogens, including acute bee paralysis virus, phorid flies, Kashmir bee virus, and Aspergillus flavus, had low infection rates that gradually declined during the detection period. The occurrence of honeybee pathogens peaked in July. SBV was the most common pathogen in A. cerana, whereas N. ceranae was predominant in A. mellifera. This study provides information regarding the current status of honey bee pathogens and presents the trend of the occurrence of each pathogen in South Korea. These data are important for predicting outbreaks of honey bee diseases in the country.

Circular RNA ame_circ_000115 regulates expression of genes in larval gusts of Apis mellifera ligustica stressed by Ascosphaera apis

Circular RNAs (circRNAs) are a new class of non-coding RNAs, which have been confirmed to regulate insect gene expression and immune response through multiple manners such as competing endogenous RNA (ceRNA) regulatory network. Currently, function of circRNA in honey bee immune response remains unclear. In this study, PCR and Sanger sequencing were performed to validate the back splicing (BS) site of ame_circ_000115 (in short ac115). RT-qPCR was used to detect the expression profile of ac115 in larval guts of Apis mellifera ligustica stressed by Ascosphaera apis. Dual-luciferase reporter gene assay was conducted to verify the binding relationship between ac115 and ame-miR-13b. Interference of ac115 in larval guts was carried out by feeding specific siRNA, followed by determination of the effect of ac115 interference on expression of six genes relevant to host immune response. The results confirmed the existence of BS site within ac115. Compared with the un-inoculated group, the expression of ac115 in 4-day-old larval gut of the A. apis-inoculated group was up-regulated with extreme significance (P < 0.000 1), while that in 5- and 6-day-old larval guts were significantly up-regulated (P < 0.05). The brightness of specific band for ac115 in 4-, 5- and 6-day-old larval guts of the siRNA-circ_000115-fed group gradually became weak, whereas that of the siRNA-scrambl-fed group was pretty high without obvious variation. Compared with that of the siRNA-scramble-fed group, the expression of ac115 in 4-day-old larval gut of the siRNA-circ_000115-fed group was significantly down-regulated (P < 0.05), whereas that of the 5- and 6-day-old larval guts were down-regulated with extreme significance (P < 0.001). Ame-miR-13b was truly existed and expressed in A. m. ligustica larval guts, and there was true binding relationship between ac115 and ame-miR-13b. Compared with that of the siRNA-scramble-fed group, the expression of antimicrobial peptide genes hymenoptaecin and abaecin in 6-day-old larval gut of the siRNA-circ_000115-fed group was significantly up-regulated (P < 0.05), while that of ecdysone receptor (Ecr) was down-regulated with extreme significance (P < 0.01). These results indicate that ac115 is truly expressed in A. m. ligustica larval guts, BS site truly exists within ac115, and effective interference of ac115 in A. m. ligustica larval guts can be achieved via feeding siRNA. Moreover, ac115 potentially regulates Ecr expression through adsorption of ame-miR-13b and expression of hymenoptaecin and abaecin using a non-ceRNA manner, further participating in host stress-response.

Nano-managing silver and zinc as bio-conservational approach against pathogens of the honey bee

Herein, silver and zinc oxide Nanoparticles (NPs) were synthesized by using W. coagulant fruit extract as reducing agent and capping agent. The green synthesized NP with distinct properties were used for novel application against fungal and bacterial pathogen of honey bee (A. mellifera). The UV-spectroscopy confirms the synthesis of silver and zinc oxide NPs at 420 nm and 350 nm respectively. Further, XRD evaluated the monoclinic structure of Ag NPs while ZnO NPs showed wurtzite hexagonalcrystlized structure. Resistant honey bee pathogens such Paenibacilluslarvae, Melissococcus plutonius and Ascosphaera apis were isolated, identified and cultured in vitro to assess the antimicrobial potentials of Ag and ZnO NPs. Additionally, different biomolecules provide access to achieve maximum and stable Ag and ZnO NPs. It was also observed that with increasing the concentration of zinc oxide NPs and sliver NPs, zone of inhibition was also increased. Thus, present findings show that plant extracts can be a useful natural resource to prepare functional nonmaterial for targeted applications especially in the field of apicultural research.

Effect of Ascosphaera apis Infestation on the Activities of Four Antioxidant Enzymes in Asian Honey Bee Larval Guts.

Ascosphaera apis infects exclusively bee larvae and causes chalkbrood, a lethal fungal disease that results in a sharp reduction in adult bees and colony productivity. However, little is known about the effect of A. apis infestation on the activities of antioxidant enzymes in bee larvae. Here, A. apis spores were purified and used to inoculate Asian honey bee (Apis cerana) larvae, followed by the detection of the host survival rate and an evaluation of the activities of four major antioxidant enzymes. At 6 days after inoculation (dpi) with A. apis spores, obvious symptoms of chalkbrood disease similar to what occurs in Apis mellifera larvae were observed. PCR identification verified the A. apis infection of A. cerana larvae. Additionally, the survival rate of larvae inoculated with A. apis was high at 1-2 dpi, which sharply decreased to 4.16% at 4 dpi and which reached 0% at 5 dpi, whereas that of uninoculated larvae was always high at 1~8 dpi, with an average survival rate of 95.37%, indicating the negative impact of A. apis infection on larval survival. As compared with those in the corresponding uninoculated groups, the superoxide dismutase (SOD) and catalase (CAT) activities in the 5- and 6-day-old larval guts in the A. apis-inoculated groups were significantly decreased (p &lt; 0.05) and the glutathione S-transferase (GST) activity in the 4- and 5-day-old larval guts was significantly increased (p &lt; 0.05), which suggests that the inhibition of SOD and CAT activities and the activation of GST activity in the larval guts was caused by A. apis infestation. In comparison with that in the corresponding uninoculated groups, the polyphenol oxidase (PPO) activity was significantly increased (p &lt; 0.05) in the 5-day-old larval gut but significantly reduced (p &lt; 0.01) in the 6-day-old larval gut, indicating that the PPO activity in the larval guts was first enhanced and then suppressed. Our findings not only unravel the response of A. cerana larvae to A. apis infestation from a biochemical perspective but also offer a valuable insight into the interaction between Asian honey bee larvae and A. apis.

Ame-miR-34 Modulates the Larval Body Weight and Immune Response of Apis mellifera Workers to Ascosphara apis Invasion.

MiRNAs are critical regulators of numerous physiological and pathological processes. Ascosphaera apis exclusively infects bee larvae and causes chalkbrood disease. However, the function and mechanism of miRNAs in the bee larval response to A. apis infection is poorly understood. Here, ame-miR-34, a previously predicted miRNA involved in the response of Apis mellifera larvae to A. apis invasion, was subjected to molecular validation, and overexpression and knockdown were then conducted to explore the regulatory functions of ame-miR-34 in larval body weight and immune response. Stem-loop RT-PCR and Sanger sequencing confirmed the authenticity of ame-miR-34 in the larval gut of A. mellifera. RT-qPCR results demonstrated that compared with that in the uninfected larval guts, the expression level of ame-miR-34 was significantly downregulated (p < 0.001) in the guts of A. apis-infected 4-, 5-, and 6-day-old larvae, indicative of the remarkable suppression of host ame-miR-34 due to A. apis infection. In comparison with the corresponding negative control (NC) groups, the expression level of ame-miR-34 in the larval guts in the mimic-miR-34 group was significantly upregulated (p < 0.001), while that in the inhibitor-miR-34 group was significantly downregulated (p < 0.01). Similarly, effective overexpression and knockdown of ame-miR-34 were achieved. In addition, the body weights of 5- and 6-day-old larvae were significantly increased compared with those in the mimic-NC group; the weights of 5-day-old larvae in the inhibitor-miR-34 group were significantly decreased in comparison with those in the inhibitor-NC group, while the weights of 4- and 6-day-old larvae in the inhibitor-miR-34 group were significantly increased, indicating the involvement of ame-miR-34 in modulating larval body weight. Furthermore, the expression levels of both hsp and abct in the guts of A. apis-infected 4-, 5-, and 6-day-old larvae were significantly upregulated after ame-miR-34 overexpression. In contrast, after ame-miR-34 knockdown, the expression levels of the aforementioned two key genes in the A. apis-infected 4-, 5-, and 6-day-old larval guts were significantly downregulated. Together, the results demonstrated that effective overexpression and knockdown of ame-miR-34 in both noninfected and A. apis-infected A. mellifera larval guts could be achieved by the feeding method, and ame-miR-34 exerted a regulatory function in the host immune response to A. apis invasion through positive regulation of the expression of hsp and abct. Our findings not only provide a valuable reference for the functional investigation of bee larval miRNAs but also reveal the regulatory role of ame-miR-34 in A. mellifera larval weight and immune response. Additionally, the results of this study may provide a promising molecular target for the treatment of chalkbrood disease.

Unveiling the circRNA-Mediated Immune Responses of Western Honey Bee Larvae to Ascosphaera apis Invasion.

Western honey bee (Apis mellifera), a eusocial insect with a superior economic and ecological value, is widely used in the beekeeping industry throughout the world. As a new class of non-coding RNAs (ncRNAs), circular RNAs (circRNAs) participate in the modulation of considerable biological processes, such as the immune response via diverse manners. Here, the identification, characteristic investigation, and molecular verification of circRNAs in the Apis mellifera ligustica larval guts were conducted, and the expression pattern of larval circRNAs during the Ascosphaera apis infection was analyzed, followed by the exploration of the potential regulatory part of differentially expressed circRNAs (DEcircRNAs) in host immune responses. A total of 2083 circRNAs in the larval guts of A. m. ligustcia were identified, with a length distribution ranging from 106 nt to 92,798 nt. Among these, exonic circRNAs were the most abundant type and LG1 was the most distributed chromosome. Additionally, 25, 14, and 30 up-regulated circRNAs as well as 26, 25, and 62 down-regulated ones were identified in the A. apis-inoculated 4-, 5-, and 6-day-old larval guts in comparison with the corresponding un-inoculated larval guts. These DEcircRNAs were predicted to target 35, 70, and 129 source genes, which were relative to 12, 23, and 20 GO terms as well as 11, 10, and 27 KEGG pathways, including 5 cellular and humoral immune pathways containing apoptosis, autophagy, endocytosis, MAPK, Toll, and Imd signaling pathways. Furthermore, complex competing endogenous RNA (ceRNA) regulatory networks were detected to be formed among DEcircRNAs, DEmiRNAs, and DEmRNAs. The Target DEmRNAs were engaged in 24, 20, and 25 functional terms as well as 62, 80, and 159 pathways, including several vital immune defense-associated pathways, namely the lysosome, endocytosis, phagosome, autophagy, apoptosis, MAPK, Jak-STAT, Toll, and Imd signaling pathways. Finally, back-splicing sites within 15 circRNAs and the difference in the 9 DEcircRNAs' expression between un-inoculated and A. apis-inoculated larval guts were confirmed utilizing molecular methods. These findings not only enrich our understanding of bee host-fungal pathogen interactions, but also lay a foundation for illuminating the mechanism underlying the DEcircRNA-mediated immune defense of A. m. ligustica larvae against A. apis invasion.

Comprehensive investigation and regulatory function of lncRNAs engaged in western honey bee larval immune response to Ascosphaera apis invasion.

Ascosphaera apis is a fungal pathogen that exclusively infects bee larvae, causing chalkbrood disease, which results in severe damage for beekeeping industry. Long non-coding RNAs (lncRNAs) are versatile regulators in various biological processes such as immune defense and host-pathogen interaction. However, expression pattern and regulatory role of lncRNAs involved in immune response of bee host to A. apis invasion is still very limited. Here, the gut tissues of Apis mellifera ligustica 4-, 5-, and 6-day-old larvae inoculated by A. apis spores (AmT1, AmT2, and AmT3 groups) and corresponding un-inoculated larval guts (AmCK1, AmCK2, and AmCK3 groups) were prepared and subjected to deep sequencing, followed by identification of lncRNAs, analysis of differentially expressed lncRNAs (DElncRNAs), and investigation of competing endogenous RNA (ceRNA) network. In total, 3,746 A. m. ligustica lncRNAs were identified, including 78 sense lncRNAs, 891 antisense lncRNAs, 1,893 intergenic lncRNAs, 346 bidirectional lncRNAs, and 210 intronic lncRNAs. In the 4-, 5-, and 6- comparison groups, 357, 236, and 505 DElncRNAs were discovered. Additionally, 217, 129, and 272 DElncRNAs were respectively predicted to regulate neighboring genes via cis-acting manner, and these targets were associated with a series of GO terms and KEGG pathways of great importance, such as response to stimulus and Jak-STAT signaling pathway. Moreover, 197, 95, and 356 DElncRNAs were observed to target 10, eight, and 21 DEmiRNAs and further target 147, 79, and 315 DEmRNAs, forming complex regulatory networks. Further investigation suggested that these targets were engaged in several key cellular and humoral immune pathways, such as phagosome and MAPK signaling pathway. Ultimately, the expression trends of nine randomly selected DElncRNAs were verified by RT-qPCR, confirming the authenticity and reliability of our transcriptome data. Findings in this current work not only provide candidate DElncRNAs for functional study, but also lay a foundation for unclosing the mechanism underlying DElncRNA-regulated larval immune responses to A. apis invasion.

Microbiological and chemical characterization of water kefir: An innovative source of potential probiotics for bee nutrition

We evaluated the microbial composition of water kefir grains and beverage over the course of one year to determine whether the number and type of microorganisms changed over the time. Bacteria and yeast colonies with different morphologies were isolated from water kefir and their antimicrobial activity was evaluated against Paenibacillus larvae and Ascosphaera apis. A chemical characterization of kefir was also carried out. Our results confirmed that bacteria and yeasts were more numerous in kefir grains compared with those in the beverage. The counts of microorganisms declined, although an important microbial community was still present in kefir after the long storage period. Eleven strains which inhibited bee pathogens were isolated from kefir. Genotypic results demonstrated that these isolates included Lentilactobacillus hilgardii, Lentilactobacillus buchneri and Saccharomyces cerevisiae. Thus, water kefir may be an innovative source of potential probiotic strains for bee nutrition in order to control honeybee diseases.

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.

Physicochemical properties, molecular structure, antioxidant activity, and biological function of extracellular melanin from Ascosphaera apis.

Ascosphaera apis spores containing a dark-colored pigment infect honeybee larvae, resulting in a large-scale collapse of the bee colony due to chalkbrood disease. However, little is known about the pigment or whether it plays a role in bee infection caused by A. apis. In this study, the pigment was isolated by alkali extraction, acid hydrolysis, and repeated precipitation. Ultraviolet (UV) analysis revealed that the pigment had a color value of 273, a maximum absorption peak at 195 nm, and a high alkaline solubility (7.67%) and acid precipitability. Further chemical structure analysis of the pigment, including elemental composition, Fourier transform infrared (FTIR) spectroscopy, Raman spectroscopy, mass spectrometry, and nuclear magnetic resonance (NMR), proved that it was a eumelanin with a typical indole structure. The molecular formula of melanin is C10H6O4N2, and its molecular weight is 409 Da. Melanin has hydroxyl, carboxyl, amino, and phenolic groups that can potentially chelate to metal ions. Antioxidant function analyses showed that A. apis melanin had a high scavenging activity against superoxide, hydroxyl, and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals, and a high reducing ability to Fe3+. Indirect immunofluorescence assay (IFA), scanning electron microscopy (SEM), and transmission electron microscopy (TEM) analyses showed that A. apis melanin was located on the spore wall. The spore wall localization, antioxidant activity, and metal ion chelating properties of fungal melanin have been suggested to contribute to spore pathogenicity. However, further infection experiments showed that melanin-deficient spores did not reduce the mortality of bee larvae, indicating that melanin does not increase the virulence of A. apis spores. This study is the first report on melanin produced by A. apis, providing an important background reference for further study on its role in A. apis.

Two Pathogenic Fungi Isolated From Chalkbrood Samples and Honey Bee Viruses They Carried.

Ascosphaera apis and some Aspergillus species are the main pathogenic fungi of honey bee, and A. apis is the pathogen of chalkbrood disease. However, the infection mechanism of them is incompletely known and it is still unclear whether other factors impact their pathogenesis. In this study, Aspergillus tubingensis were obtained from the chalkbrood bee samples for the first time. Our results showed that A. tubingensis could promote the accumulation of the spores of A. apis. Pathogenicity test found that inoculation of the spores of the two fungi alone or their combination could induce disease characterization of chalkbrood and stonebrood but the extent was less than those in field. To further identify other pathogens impacted the pathogenesis, we found several honey bee viruses presented in the pathogenic fungi A. apis and A. tubingensis, which were different from previous reported. Our results indicated that acute bee paralysis virus (ABPV) and chronic bee paralysis virus (CBPV) could replicate in these two fungi and increased in titer with the going of cultivation time. In addition, CBPV could not only transmit vertically to the next generation by spores, but also spread horizontally to different fungi through hyphal anastomosis. These results suggested that the honey bee chalkbrood contained the other pathogenic fungi besides A. apis, the interactions between different pathogens of chalkbrood microbial communities may influence the prevalence of chalkbrood. Moreover, the discovery of honey bee viruses and their transmission mode in these two fungi enhanced the potential of exploring fungi virus as valuable factors that cause fungal disease outbreak.

The inhibitory action of plant extracts on the mycelial growth of Ascosphaera apis, the causative agent of chalkbrood disease in Honey bee

Ascosphaera apis is a fungal pathogen, which causes chalkbrood disease in bees and is threatening beekeeping worldwide. The demand for organic honey for export has lately heightened hence the biological control is the option. This study aimed at the in vitro evaluation of the potency of plant extracts against chalkbrood disease for the possibility of being employed as a biological control strategy. The results showed that the combination of plant extracts from cinnamon with spearmint, cinnamon with lemongrass, cinnamon with geranium, and cinnamon with palmarosa at a concentration of 25% and 12.5% inhibited mycelial growth of A. apis by 100%. This demonstrated the potentiality of combining different plant extracts in controlling this disease. In addition, oregano caused inhibition of up to 100% singly. Conclusively, cinnamon in combination with several extracts has a great potential in curbing this disease while oregano offers an amazing remedy and hence the best formulations should be generated for the beekeeper to utilize.