Ascosphaera Apis Research Articles

Study of the chemical composition of the essential oil derived from Thymus lancéolatus desf, an endemic aromatic plant native from Algeria and evaluation of its efficacy against Paenibacillus larvae and Ascosphaera Apis, two diseases affecting the honeybees, Apis mellifera

The essential oil and hydrolate of Thymus lancéolatus desf, an endemic aromatic plant native to the mountains of northwestern Algeria, were tested in vitro for their antimicrobial activity against the bacteria Paenibacillus larvae, the causative agent of American foulbrood, and the fungi Ascosphaera apis, the responsible agent of chalkbrood disease in honeybees, Apis mellifera. The oil was extracted from various aerial parts of the plant using steam distillation. It shows high efficacy against both diseases, while its hydrolat demonstrates high efficacy against A.apis alone. The lethal doses (LD100) of the fungi were estimated at 0.034% and 25.30% for the essential oil and the hydrolat, respectively. The estimated concentration of the DL100 bacteria was 17.37 ul/Disc of EO. The hydrolate had no effect on P. larvae. This exceptional double efficacy of T.lancéolatus desf EO has been attributed to its particular active compound, Durenol, which constitutes 34.8% of the oil and the high quantities of its precursors (γ-Terpinene (16.71%) and Sylvestrene (15.17%) contribute to its effectiveness. Also, the abcence of acetate compound further enhances its efficacy. EO of Thyme was proven to be the most effective essential oil in vitro against P. larvae and A. apis, making it an excellent natural alternative to antibiotics.

Chalkbrood Disease Caused by Ascosphaera apis in Honey Bees (Apis mellifera)-Morphological and Histological Changes in Infected Larvae.

Chalkbrood is a mycological brood disease of the Western honey bee (Apis mellifera), caused by the fungus Ascosphaera apis. The aim of this study was the investigation of the pathology of artificially reared Apis mellifera larvae, experimentally infected with A. apis spores (1.0 × 103 spores/larva). Non-infected larvae served as control. Five living larvae and every dead larva were collected daily (day 1-7 p.i.). All larvae were macroscopically measured, photographed, formalin-fixed, and histologically processed (hematoxylin-eosin stain, Grocott silvering). Histological sections were digitized, and the size of the larvae was measured (mouth-after length, area) and statistically analyzed. Twenty-six larvae from the collected larvae (n = 64; 23 dead, 3 alive) showed histological signs of infection from 3 d p.i. onwards. The dead larvae showed macroscopically white/brown deposits, indistinct segmentation, and a lack of body elongation. Infected larvae were significantly smaller than the controls on days 3 p.i. (p < 0.05), 4 p.i. (p < 0.001), and 6 p.i. (p < 0.05). The early time of death, the low number of transitional stages, and the strong penetration of the larval carcass with fungal mycelium indicate a rapid and fulminant infection process, which is probably relevant for spreading the disease within the colony.

Morphological and Molecular Identification and Mating Type Detection of Chalkbrood Fungal Pathogen &lt;i&gt;Ascosphaera apis&lt;/i&gt; in &lt;i&gt;Apis mellifera&lt;/i&gt; L. in Southern India

Chalkbrood Ascosphaera apis is a fungal brood pathogen that exerts considerable biotic stress on honey bees worldwide. Chalkbrood was noticed at Coimbatore in Apis mellifera colonies for the first time. Precise identification of the fungal pathogen and their mating types is crucial to develop effective disease management strategies. Hence, the present study aimed to isolate and identify fungal pathogen morphologically and genomically. Scanning Electron Microscopic (SEM) analysis was done and confirmed the essential morphological characteristics of the isolated fungus such as spore cysts (51-86 µm in diameter), spore balls (8-15 µm in diameter), and ascospores (1.7-2.6 µm in length). Molecular characterization using internal transcribed sequence (ITS)-PCR of the fungal isolate indicated 99-100% sequence similarity to A. apis. In addition, the multiplex PCR assay was performed and the mating types MAT1-1 and MAT1-2 were successfully detected and named TNAU CBD MAT1 and TNAU CBD MAT2, respectively.

Effect of Media on the Mycelial Growth of Honey Bee Fungal Pathogen &lt;i&gt;Ascosphaera apis&lt;/i&gt; Mating Types

The fungal pathogen Ascosphaera apis significantly impacts the honey bee Apis mellifera by causing larval mortality and reducing colony productivity. Understanding the growth dynamics of A. apis under different media conditions is crucial for advancing research on its biology and pathogenicity. This study evaluates the effect of eight artificial media namely Potato Dextrose Agar (PDA), Carrot Dextrose Agar (CDA), Beetroot Dextrose Agar (BDA), Tender Coconut Water (TCW) media, Sabouraud Dextrose Agar (SDA), Sabouraud Dextrose Agar Yeast (SDAY), Sabouraud Maltose Agar Yeast (SMAY), and Malt Extract Agar (MEA) on the mycelial growth and reproductive structure formation of A. apis's two mating types. Results indicated significant differences in mycelial growth across media. SDA (75.40 mm dia) and SDAY (74.83 mm dia) supported maximum growth for mating type 1 (MAT1) and, SDA (72.72 mm dia) and BDA (68.17 mm dia) yielded the maximum growth for mating type 2 (MAT2). The TCW resulted in the least growth for MAT1 (44.19 mm dia) and MAT2 (41.25 mm dia). The growth patterns showed that media supplemented with sugars generally enhanced A. apis growth, whereas non-sugar media did not. Furthermore, reproductive structures formed faster on PDA and CDA than on other media. The study confirms that media composition, particularly sugar content, critically influences the growth and development of A. apis, providing insights that could help to develop better management strategies for controlling chalkbrood disease in honey bee colonies.

Occurrence of Nosema ceranae, Ascosphaera apis and trypanosomatids in Vespa orientalis linneus 1771

Vespa orientalis is spreading across the Italian and European territories leading to new interactions among species, which could lead to the transmission of pathogens between species. Detection of honey bee viruses in V. orientalis has already been revealed in both adults and larvae, while no information is available regarding parasitic occurrence. Sixty adult hornets collected across apiaries in the South of Italy were subjected to cytological, histopathological and biomolecular examination to evaluate the occurrence of Nosema ceranae, Ascosphaera apis, Lotmaria passim, Crithidia mellificae, and Crithidia bombi.Cytological examination revealed the presence of Nosema spores in 38.33% of individuals while histopathological analysis showed the presence of L. passim-like elements in the rectum of two examined specimens and the presence of fungal hyphae in the small intestine of another hornet. Biomolecular investigation revealed that N. ceranae was the most prevalent pathogen (50.0%), followed by A. apis (6.66%), L. passim (6.66%) and C. bombi (6.0%).

Pollen products collected from honey bee hives experiencing minor stress have altered fungal communities and reduced antimicrobial properties.

Fungi are increasingly recognised to play diverse roles within honey bee hives, acting as pathogens, mutualists, and commensals. Pollen products, essential for hive nutrition, host significant fungal communities with potential protective and nutritional benefits. In this study, we profile the fungal communities and antifungal properties of three pollen products from healthy and stressed hives: fresh pollen collected by forager bees from local plants; stored pollen packed into the comb inside the hive; and bee bread, which is stored pollen following anaerobic fermentation used for bee and larval nutrition. Using amplicon sequencing, we found significant differences in fungal community composition, with hive health and sample type accounting for 8.8% and 19.3% of variation in beta diversity, respectively. Pollen and bee bread extracts had species-specific antimicrobial activity and inhibited the fungal hive pathogens Ascosphaera apis, Aspergillus flavus, and Aspergillus fumigatus, and the bacterial hive pathogen Paenibacillus larvae. Activity was positively correlated with phenolic and antioxidant content and was diminished in stressed hives. The plant source of pollen determined by amplicon sequencing differed in stressed hives, suggesting altered foraging behaviour. These findings illustrate the complex interplay between honey bees, fungal communities, and hive products, which should be considered in hive management and conservation.

Micronutrient Deficiency May Be Associated with the Onset of Chalkbrood Disease in Honey Bees.

Chalkbrood is a disease of honey bee brood caused by the fungal parasite Ascosphaera apis. Many factors such as genetics, temperature, humidity and nutrition influence the appearance of clinical symptoms. Poor nutrition impairs the immune system, which favors the manifestation of symptoms of many honey bee diseases. However, a direct link between dietary ingredients and the symptoms of chalkbrood disease has not yet been established. We show here that the elemental composition of chalkbrood mummies and healthy larvae from the same infected hives differ, as well as that mummies differ from larvae from healthy hives. Chalkbrood mummies had the highest concentration of macroelements such as Na, Mg, P, S, K and Ca and some microelements such as Rb and Sn, and at the same time the lowest concentration of B, As, Sr, Ag, Cd, Sb, Ba and Pb. Larvae from infected hives contained less Pb, Ba, Cs, Sb, Cd, Sr, As, Zn, Cu, Ni, Co, Mn, Cr, V and Al in contrast to healthy larvae from a disease-free apiary. This is the first study to demonstrate such differences, suggesting that an infection alters the larval nutrition or that nutrition is a predisposition for the outbreak of a chalkbrood infection. Though, based on results obtained from a case study, rather than from a controlled experiment, our findings stress the differences in elements of healthy versus diseased honey bee larvae.

Transcriptional dynamics and regulatory function of milRNAs in Ascosphaera apis invading Apis mellifera larvae.

In the present study, small RNA (sRNA) data from Ascosphaera apis were filtered from sRNA-seq datasets from the gut tissues of A. apis-infected Apis mellifera ligustica worker larvae, which were combined with the previously gained sRNA-seq data from A. apis spores to screen differentially expressed milRNAs (DEmilRNAs), followed by trend analysis and investigation of the DEmilRNAs in relation to significant trends. Additionally, the interactions between the DEmilRNAs and their target mRNAs were verified using a dual-luciferase reporter assay. In total, 974 A. apis milRNAs were identified. The first base of these milRNAs was biased toward U. The expression of six milRNAs was confirmed by stem-loop RT-PCR, and the sequences of milR-3245-y and milR-10285-y were validated using Sanger sequencing. These miRNAs grouped into four significant trends, with the target mRNAs of DEmilRNAs involving 42 GO terms and 120 KEGG pathways, such as the fungal-type cell wall and biosynthesis of secondary metabolites. Further investigation demonstrated that 299 DEmilRNAs (novel-m0011-3p, milR-10048-y, bantam-y, etc.) potentially targeted nine genes encoding secondary metabolite-associated enzymes, while 258 (milR-25-y, milR-14-y, milR-932-x, etc.) and 419 (milR-4561-y, milR-10125-y, let-7-x, etc.) DEmilRNAs putatively targeted virulence factor-encoded genes and nine genes involved in the MAPK signaling pathway, respectively. Additionally, the interaction between ADM-B and milR-6882-x, as well as between PKIA and milR-7009-x were verified. Together, these results not only offer a basis for clarifying the mechanisms underlying DEmilRNA-regulated pathogenesis of A. apis and a novel insight into the interaction between A. apis and honey bee larvae, but also provide candidate DEmilRNA-gene axis for further investigation.

Probiotic Potential of Bacillus Subtilis Strain I3: Antagonistic Activity Against Chalkbrood Pathogen and Pesticide Degradation for Enhancing Honeybee Health.

To explore the potential of probiotic candidates beneficial for honeybee health through the modulation of the gut microbiome, bee gut microbes were isolated from bumblebee (Bombus terrestris) and honeybee (Apis mellifera) using diverse media and cultural conditions. A total of 77 bee gut bacteria, classified under the phyla Proteobacteria, Firmicutes, and Actinobacteria, were identified. The antagonistic activity of the isolates against Ascosphaera apis, a fungal pathogen responsible for chalkbrood disease in honeybee larvae, was investigated. The highest growth inhibition percentage against A. apis was demonstrated by Bacillus subtilis strain I3 among the bacterial strains. The presence of antimicrobial peptide genes in the I3 strain was detected using PCR amplification of gene fragments encoding surfactin and fengycin utilizing specific primers. The export of antimicrobial peptides by the I3 strain into growth medium was verified using liquid chromatography coupled with mass spectroscopy. Furthermore, the strain's capabilities for degrading pesticides, used for controlling varroa mites, and its spent growth medium antioxidant activity were substantiated. The survival rate of honeybees infected with (A) apis was investigated after feeding larvae with only medium (fructose + glucose + yeast extract + royal jelly), (B) subtilis I3 strain, A. apis with medium and I3 strain + A. apis with medium. Honeybees receiving the I3 strain + A. apis exhibited a 50% reduction in mortality rate due to I3 strain supplementation under experimental conditions, compared to the control group. In silico molecular docking revealed that fengycin hydrolase from I3 strain effectively interacted with tau-fluvalinate, suggesting its potential in bee health and environmental protection. Further studies are needed to confirm the effects of the I3 strain in different populations of honey bees across several regions to account for genetic and environmental variations.

Dynamics and regulatory role of circRNAs in Asian honey bee larvae following fungal infection

Non-coding RNA (ncRNA) plays a vital part in the regulation of immune responses, growth, and development in plants and animals. Here, the identification, characteristic analysis, and molecular verification of circRNAs in Apis cerana cerana worker larval guts were conducted, followed by in-depth investigation of the expression pattern of larval circRNAs during Ascosphaera apis infection and exploration of the potential regulatory part of differentially expressed circRNAs (DEcircRNAs) in host immune responses. A total of 3178 circRNAs in the larval guts of A. c. cerana were identified, with a length distribution ranging from 15 to 96,007 nt. Additionally, 155, 95, and 86 DEcircRNAs were identified in the in the 4-, 5-, and 6-day-old larval guts following A. apis infection. These DEcircRNAs were predicted to target 29, 25, and 18 parental genes relevant to 12, 20, and 17 GO terms as well as 144, 114, and 61 KEGG pathways, including 5 cellular and 4 humoral immune pathways. Complex competing endogenous RNA (ceRNA) regulatory networks were detected as being formed among DEcircRNAs, DEmiRNAs, and DEmRNAs. The target DEmRNAs were engaged in 36, 47, and 47 GO terms as well as 331, 332, and 331 pathways, including 6 cellular and 6 humoral immune pathways. Further, 19 DEcircRNAs, 5 DEmiRNAs, and 3 mRNAs were included in the sub-networks relative to 3 antioxidant enzymes. Finally, back-splicing sites within 15 circRNAs and the difference in the 15 DEcircRNAs’ expression between uninoculated and A. apis–inoculated larval guts were confirmed based on 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. c. cerana larvae against A. apis invasion.Key points• The expression pattern of circRNAs was altered in the A. cerana worker larval guts following A. apis infection.• Back-splicing sites within 15 A. cerana circRNAs were verified using molecular approaches. DEcircRNAs potentially modulated immune responses and antioxidant enzymes in A. apis–challenged host guts.

Ecological and social factors influence interspecific pathogens occurrence among bees

The interspecific transmission of pathogens can occur frequently in the environment. Among wild bees, the main spillover cases are caused by pathogens associated with Apis mellifera, whose colonies can act as reservoirs. Due to the limited availability of data in Italy, it is challenging to accurately assess the impact and implications of this phenomenon on the wild bee populations. In this study, a total of 3372 bees were sampled from 11 Italian regions within the BeeNet project, evaluating the prevalence and the abundance of the major honey bee pathogens (DWV, BQCV, ABPV, CBPV, KBV, Nosema ceranae, Ascosphaera apis, Crithidia mellificae, Lotmaria passim, Crithidia bombi). The 68.4% of samples were positive for at least one pathogen. DWV, BQCV, N. ceranae and CBPV showed the highest prevalence and abundance values, confirming them as the most prevalent pathogens spread in the environment. For these pathogens, Andrena, Bombus, Eucera and Seladonia showed the highest mean prevalence and abundance values. Generally, time trends showed a prevalence and abundance decrease from April to July. In order to predict the risk of infection among wild bees, statistical models were developed. A low influence of apiary density on pathogen occurrence was observed, while meteorological conditions and agricultural management showed a greater impact on pathogen persistence in the environment. Social and biological traits of wild bees also contributed to defining a higher risk of infection for bivoltine, communal, mining and oligolectic bees. Out of all the samples tested, 40.5% were co-infected with two or more pathogens. In some cases, individuals were simultaneously infected with up to five different pathogens. It is essential to increase knowledge about the transmission of pathogens among wild bees to understand dynamics, impact and effects on pollinator populations. Implementing concrete plans for the conservation of wild bee species is important to ensure the health of wild and human-managed bees within a One-Health perspective.

Bacterial Strains Isolated from Stingless Bee Workers Inhibit the Growth of Apis mellifera Pathogens.

Apis mellifera bees are an important resource for the local economy of various regions in Argentina and the maintenance of natural ecosystems. In recent years, different alternatives have been investigated to avoid the reduction or loss of colonies caused by pathogens and parasites such as Ascosphaera apis, Aspergillus flavus, and Paenibacillus larvae. We focused on bacterial strains isolated from the intestine of native stingless bees, to elucidate their antagonistic effect on diseases of A. mellifera colonies. For this purpose, worker bees of the species Tetragonisca fiebrigi, Plebeia spp., and Scaptotrigona jujuyensis were captured from the entrance to tree hives and transported to the laboratory, where their intestines were extracted. Twenty bacterial colonies were isolated from the intestines, and those capable of inhibiting enterobacteria in vitro and producing organic acids, proteases, and chitinases were selected. Four genera, Levilactobacillus, Acetobacter, Lactiplantibacillus, and Pantoea, were selected and identified by the molecular marker that codes for the 16S rRNA gene. For inhibition assays, cell suspensions and cell-free suspensions were performed. All treatments showed significant antibacterial effects, in comparison with the controls, against P. larvae and antifungal effects against A. apis and A. flavus. However, the mechanisms by which these bacteria inhibit the growth of these pathogens were not studied.

Atmospheric non-thermal plasma inactivation of Ascosphaera apis, the causative agent of chalkbrood disease in honeybee

Ascosphaera apis is a worldwide pathogenic fungi of honeybees that can cause a decline in bee populations. In this study, we investigated the antifungal activity of non-thermal plasma on fungal growth. Spore inactivation after exposure to gas plasma by liquid phase and plasma activated water (PAW) and pathogenicity of A. apis in vivo were also examined. The results demonstrated that the mycelial growth of fungi was completely inhibited after argon plasma treatment. Both gas plasma and PAW exposures resulted in a significant decrease of A. apis spore numbers, maximum reduction of 1.71 and 3.18-fold, respectively. Germinated fungal spores on potato dextrose agar were also reduced after plasma treatment. SEM analysis revealed a disruption in the morphological structure of the fungal spores. The pathogenicity of A. apis on honeybee larvae was decreased after spores treated by gas plasma and PAW with a disease inhibition of 63.61 ± 7.28% and 58.27 ± 5.87%, respectively after 7 days of cultivation. Chalkbrood in honey bees have limited control options and our findings are encouraging. Here, we demonstrate a possible alternative control method using non-thermal plasma for chalkbrood disease in honeybees.

IMPROVEMENT OF TECHNOLOGICAL STAGES IN BEEKEEPING WITH DISINFECTION MEASURES

Disinfection is a very important technological measure, which is carried out to combat infectious diseases of bees and prevent contamination of beekeeping products with harmful microorganisms. The disinfection procedure is very complex, and its effectiveness depends on a large number of factors: the spectrum of the disinfectant, the method of application, the concentration of the disinfectant, the exposure time, and the properties of the environment to be disinfected. Active components of disinfectants usually affect the metabolism of microorganisms. For disinfection in beekeeping, we can use iodine preparations, bases, acids, quaternary ammonium compounds, oxidizers (hydrogen peroxide, peracetic acid). Effective chemical disinfectants are a warm solution of sodium hydroxide, oxidizers, so these disinfectants are most often used in beekeeping. However, the negative properties of these disinfectants should also be considered. There is no perfect disinfectant. Because of the toxicity and other harmful effects of disinfectants, and often their ineffectiveness, physical methods of disinfection should be implemented whenever possible. The article presents the results of studies of such disinfectants as Septox, PHMG – HC and Crystal-700 in concentrations of 0.25%, 0.5%, 1.5%, as well as a comparative description of their effectiveness during “in vitro” disinfection on test objects (glass, veneer, wax) contaminated with spores of Paenibacillus larvae subsp. larvae and Ascosphaera apis. Test controls were: No. 1 – sterile physiological solution; No. 2 – Desvax (4% formaldehyde solution with 0.05% DMSO), a drug approved for disinfection of bee inventory in Ukraine (Deklaratsiinyi patent 61387A, 2003, Rudenko, 2012). The test objects were immersed in the test solutions and kept for 3, 9, 18 hours. To determine the intestinal toxicity, the test substances were fed to bees and the time and number of dead individuals were monitored. According to the results of the sanitary and hygienic assessment of individual indicators of chemical disinfectants, the best disinfecting properties were demonstrated by 1.5% solutions of polyhexamethyleneguanidine hydrochloride, sodium hypochlorite and Crystal-700, which are environmentally safe, non-toxic, non-volatile, non-aggressive towards metals, plastics, wood, convenient to use. They can be recommended for long-term antiseptic protection, preventive, forced, current and final disinfection for bacterial, fungal and viral infections.

Detection of Ascosphaera apis, causing chalkbrood disease in the colonies of European honey bee, Apis mellifera in West Bengal, India

The decline of honey bee (Apis mellifera L.) populations is of great concern around the world. Among the several key drivers, dissemination of pests and pathogens is potential one. Chalkbrood is very common fungal disease of honey bee, caused due to Ascosphaera apis. In the present study, survey was conducted regarding the prevalence of diseases in A. mellifera beekeeping in Gangetic plains of West Bengal, India. Results confirmed the occurrence of chalkbrood disease in different apiaries, where dead and mummified bee larvae with cotton like chalky white or greyish-black covering were found as physical symptoms of the disease. From three surveyed apiaries, a total of 16 hives out of 113 hives were found to be infected with the diseases; and 46 frames out of 132 frames from the infected 16 hives were found to be affected by the pathogen. Microscopic examination reflected that nearly 87% of the samples collected from the infected frame were found to be positive for the spore of Ascosphaera. The fungus associated with the disease was isolated on Potato Dextrose Agar medium, pure cultured and its genomic DNA was isolated to perform PCR and based on 18s rDNA sequencing by using specific primer pair of ITS-1 and ITS-4, the fungus was identified as Ascosphaera apis. &#x0D; Keywords: 18s rDNA, Bees, Necrotrophs, Phylogeny

Review on Chalkbrood Disease of Honey Bee

Ascosphaera apis is the sole source of the invasive mycosis known as “chalkbrood disease”, which only affects honeybee larvae. Around 5-37% less honey is produced globally as a result of the illness, and 80% of broods perish. Because of the disease’s current severity, more pesticides are used, and colonies are frequently transported over great distances, which may have created new opportunities for Ascosphaera apis infection. There are signs that the incidence of this disease, which is now widespread worldwide, may be increasing. It is also highly unlikely that Ascosphaera apis will ever be completely eradicated due to its widespread distribution throughout the world, the fact that it can persist for more than 15-years in soil near infected apiaries, and the fact that viable spores can be found in stored honey, pollen, pollen capsules and tablets, used hive components, and used beekeeping tools and equipment. Safe control of the disease has been noted to be challenging, despite the fact that management techniques, medications, and the adoption of particular chalkbrood-resistant bees have all been demonstrated to have some benefits thus far. Thus, current research for a better understanding of chalkbrood will focus on genetic engineering for better fungus manipulation, molecular investigation of Ascosphaera apis pathogenesis pathways, and the potential identification of strategies to break the pathway through the identification of responsible genes for its pathogenesis. Some researchers have noted the prevalence of chalkbrood disease and linked chalkbrood diseases to bee colony losses and decreased productivity in affected bee colonies.

New insights into changing honey bee (Apis mellifera) immunity molecules pattern and fatty acid esters, in responses to Ascosphaera apis infection

Monitoring of metabolite changes could provide valuable insights into disturbances caused by an infection and furthermore, could be used to define the status of an organism as healthy or diseased and define what could be defensive elements against the infection. The present investigation conducted a gas chromatography–mass spectrometry (GC/MS) for haemolymph of larval honey bees (Apis mellifera L.) infected with the fungal pathogen Ascosphaera apis in comparison with control haemolymph non-infected insects. Results revealed that the pathogen caused a general disturbance of metabolites detected in the haemolymph of the honey bee. The majority of metabolites identified before and after infection were fatty acid esters. The disease caused an elevation in levels of methyl oleate, methyl palmitate, and methyl stearate, respectively. Further, the disease drove to the disappearance of methyl palmitoleate, and methyl laurate. Conversely, methyl linolelaidate, and ethyl oleate were identified only in infected larvae. A high reduction in diisooctyl phthalate was recorded after the infection. Interestingly, antimicrobial activities were confirmed for haemolymph of infected honey bee larvae. In spite of the presence of some previously known bioactive compounds in healthy larvae there were no antimicrobial activities.

Characterization of an α-Amylase from the Honeybee Chalk Brood Pathogen Ascosphaera apis.

The insect pathogenic fungus, Ascosphaera apis, is the causative agent of honeybee chalk brood disease. Amylases are secreted by many plant pathogenic fungi to access host nutrients through the metabolism of starch, and the identification of new amylases can have important biotechnological applications. Production of amylase by A. apis in submerged culture was optimized using the response surface method (RSM). Media composition was modeled using Box-Behnken design (BBD) at three levels of three variables, and the model was experimentally validated to predict amylase activity (R2 = 0.9528). Amylase activity was highest (45.28 ± 1.16 U/mL, mean ± SE) in media composed of 46 g/L maltose and1.51 g/L CaCl2 at a pH of 6.6, where total activity was ~11-fold greater as compared to standard basal media. The enzyme was purified to homogeneity with a 2.5% yield and 14-fold purification. The purified enzyme had a molecular weight of 75 kDa and was thermostable and active in a broad pH range (> 80% activity at a pH range of 7-10), with optimal activity at 55 °C and pH = 7.5. Kinetic analyses revealed a Km of 6.22 mmol/L and a Vmax of 4.21 μmol/mL·min using soluble starch as the substrate. Activity was significantly stimulated by Fe2+ and completely inhibited by Cu2+, Mn2+, and Ba2+ (10 mM). Ethanol and chloroform (10% v/v) also caused significant levels of inhibition. The purified amylase essentially exhibited activity only on hydrolyzed soluble starch, producing mainly glucose and maltose, indicating that it is an endo-amylase (α-amylase). Amylase activity peaked at 99.38 U/mL fermented in a 3.7 L-bioreactor (2.15-fold greater than what was observed in flask cultures). These data provide a strategy for optimizing the production of enzymes from fungi and provide insight into the α-amylase of A. apis.

Regulatory Roles of Long Non-Coding RNAs Relevant to Antioxidant Enzymes and Immune Responses of Apis cerana Larvae Following Ascosphaera apis Invasion.

Long non-coding RNAs (lncRNAs) play an essential part in controlling gene expression and a variety of biological processes such as immune defense and stress-response. However, whether and how lncRNAs regulate responses of Apis cerana larvae to Ascosphaera apis invasion has remained unclear until now. Here, the identification and structural analysis of lncRNAs in the guts of A. cerana worker larvae were conducted, and the expression profile of larval lncRNAs during the A. apis infection process was then analyzed, followed by an investigation of the regulatory roles of differentially expressed lncRNAs (DElncRNAs) in the host response. In total, 76 sense lncRNAs, 836 antisense lncRNAs, 184 intron lncRNAs, 362 bidirectional lncRNAs, and 2181 intron lncRNAs were discovered in the larval guts. Additionally, 30 known and 9 novel lncRNAs were potential precursors for 36 and 11 miRNAs, respectively. In the three comparison groups, 386, 351, and 272 DElncRNAs were respectively identified, indicating the change in the overall expression pattern of host lncRNAs following the A. apis invasion. Analysis of cis-acting effect showed that DElncRNAs in the 4-, 5-, and 6-day-old comparison groups putatively regulated 55, 30, and 20 up- and down-stream genes, respectively, which were involved in a series of crucial functional terms and pathways, such as MAPK signaling pathway, and cell process. Analysis showed that 31, 8, and 11 DElncRNAs as potential antisense lncRNAs may interact with 26, 8, and 9 sense-strand mRNAs. Moreover, investigation of the competing endogenous RNA (ceRNA) network indicated that 148, 283, and 257 DElncRNAs were putatively regulated. The expression of target genes by targeting corresponding DEmiRNAs included those associated with antioxidant enzymes and immune responses. These results suggested that DElncRNAs played a potential part in the larval guts responding to the A. apis infection through a cis-acting manner and ceRNA mechanisms. Our findings deepen our understanding of interactions between A. cerana larvae and A. apis and offer a basis for clarifying the DElncRNA-mediated mechanisms underlying the host response to fungal invasion.

Widespread distribution of honey bee-associated pathogens in native bees and wasps: Trends in pathogen prevalence and co-occurrence

Pollinators have experienced significant declines in the past decade, in part due to emerging infectious diseases. Historically, studies have primarily focused on pathogens in the Western honey bee, Apis mellifera. However, recent work has demonstrated that these pathogens are shared by other pollinators and can negatively affect their health. Here, we surveyed honey bees and 15 native bee and wasp species for 13 pathogens traditionally associated with honey bees. The native bee and wasp species included 11 species not previously screened for pathogens. We found at least one honey bee-associated pathogen in 53% of native bee and wasp samples. The most widely distributed and commonly detected pathogens were the microsporidian Nosema ceranae, the bacterium Melissococcus plutonius, and the viruses deformed wing virus and black queen cell virus. The prevalence of viruses was generally higher in honey bees than in native bees and wasps. However, the prevalence of M. plutonius and the brood fungus Ascosphaera apis was significantly higher in some native bee species than in honey bees. The data also reveal novel trends in the association between co-occurring pathogens in honey bees and native bees and wasps at the pathogen community level. These results can inform the assessment of risks that native pollinator species face from pathogen stress, and indicate that many non-viral pathogens, notably M. plutonius and N. ceranae, are far more widely distributed and commonly found in native bees and wasps than previously thought.