In vitro activity of 15-azasterol (A25822B) against chalkbrood pathogen Ascosphaera apis in the honey bee

The antifungal agent 15-azasterol A25822B was examined for effects on the growth and development of Ascosphaera apis. The minimum inhibition concentration (MIC) of azasterol against A. apis was 1 m. Growth and development of A. apis was completely controlled at this concentration. At a concentration of 0.01 m growth of A. apis was retarded and although sporocysts were formed developing spores were not be able to reach maturation. A major effect of azasterol at this low concentration was the accumulation of lipid in the hyphae, sporocysts and immature spores. In addition it caused a conformational change in mitochondria and damage to the spore membrane structure. On the basis of these results, further investigations of azasterol for the treatment of chalkbrood disease in the honey bee are warranted.Work was performed during sabbatical leave at the University of California, Davis.     

Fine structure of the sporocysts of Ascosphaera apis during development as revealed by the scanning electron microscope

The fine morphology of the sporocysts of Ascosphaera apis (Maassen ex Claussen) Olive & Spiltoir, an entomopathogenic fungus of the immature honey bee has been studied using the scanning electron microscope. During the third day of mycelial growth in the culture medium, numerous short, branched hyphae were formed. The tip of the branch-hypha gradually expanded to form immature sporocysts. The immature sporocysts contained a large number of globules of varying sizes. The walls of the immature sporocysts were finely wrinkled, becoming smooth as the sporocyst matured. Both exterior and interior surfaces of the sporocyst wall possessed numerous papillae. Some globules in the developing sporocyst began to form immature spores which aggregated to form spore balls. The fully formed spore balls were not enveloped by a membrane.     

Effects of itraconazole on the sporocysts wall of the entomopathogenic fungi Ascosphaera apis as revealed by the scanning electron microscope

The young sporocysts had a wrinkled sporocyst wall, numerous papillae on the wall surface and the wall was granular and porous. In the itraconazole-treated culture, the walls of the young sporocysts were also wrinkled, but the characteristic papillae were replaced by larger lumps which were densely packed and coated the entire surface of the sporocyst wall. The mature sporocysts walls were smooth and possessed numerous papillae. In the itraconazole-treated culture, the walls of mature sporocysts walls were also smooth but possessed densely packed larger lumps instead of papillae. At higher magnification, each of the lumps were found to consist of numerous globules. No pores were observed as they were in the normal sporocyst wall.     

蜜蜂球囊菌菌丝和孢子中环状RNA的鉴定及比较分析

[目的]本研究旨在明确蜜蜂球囊菌(Ascosphaera apis,简称球囊菌)菌丝和孢子中环状RNA(circular RNA,circRNA)的数量、种类和表达谱差异,并探讨共有circRNA、特有circRNA和差异表达circRNA (differentially expressed circRNA,DEcir...     

Lipids stimulate spore germination in the entomopathogenic ascomycete <Emphasis Type="Italic">Ascosphaera aggregata</Emphasis>

The alfalfa leafcutting bee (Megachile rotundata) is solitary and managed on a large scale for pollination of alfalfa seed crops. The bees nest in holes drilled in wood or polystyrene blocks, and their larvae are highly prone to a fungal disease called chalkbrood. The most prevalent form of chalkbrood is caused by Ascosphaera aggregata, but this ascomycete is difficult to culture. Hyphae will grow on standard fungal media, but spore germination is difficult to achieve and highly variable. We found that germination can be enhanced with oils. Lipids derived from plants and bee larvae increased germination from 50% (without oil) to 75–85% (with oil). Percent germination was significantly greater in the presence of lipids but germination was not significantly different when different oils, including mineral oil, were used. A. aggregata spores oriented along the oil--aqueous interface in the broth in a polar fashion, with swelling and germ tube formation always occurring into the aqueous portion of the broth. The other half of the spore tended to attach to a lipid droplet, where it remained, without swelling, during germ tube formation. The physical attachment of spores to the oil--aqueous interface is what most probably stimulates spore germination, as opposed to some nutritional stimulation. However, further research is needed to determine if and where the spores encounter such an interface when germinating in the host gut, where germination normally occurs.     

Long-term storage of Ascosphaera aggregata and Ascosphaera apis, pathogens of the leafcutting bee (Megachile rotundata) and the honey bee (Apis mellifera)

Survival rates of Ascosphaera aggregata and Ascosphaera apis over the course of a year were tested using different storage treatments. For spores, the storage methods tested were freeze-drying and ultra-low temperatures, and for hyphae, freeze-drying, agar slants, and two methods of ultra-low temperatures. Spores of A. aggregata and A. apis stored well at −80 °C and after freeze-drying. A. aggregata hyphae did not store well under any of the methods tested while A. apis hyphae survived well using cryopreservation. Spores produced from cryopreserved A. apis hyphae were infective. Long-term storage of these two important fungal bee diseases is thus possible.     

结合三代测序与二代测序技术揭示蜜蜂球囊菌孢子转录组的复杂性

【目的】蜜蜂球囊菌(Ascosphaera apis)是一种专性侵染蜜蜂幼虫的致死性真菌病原。本研究旨在利用PacBio单分子实时(singlemoleculereal-time,SMRT)测序技术对蜜蜂球囊菌孢子(AaS)中基因的可变剪切(alternative splicing,AS)和可变多聚腺苷酸化(alternative polyadenylation,APA)以及长链非编码RNA (long non-coding RNA,lncRNA)进行鉴定和分析,进而揭示蜜蜂球囊菌孢子中转录组的复杂性。【方法】采用Suppa软件对蜜蜂球囊菌孢子中基因的AS事件进行鉴定。通过RT-PCR对不同类型的AS事件进行验证。采用TAPIS pipeline对蜜蜂球囊菌孢子基因的APA位点进行鉴定。利用MEME软件分析孢子全长转录本的poly(A)剪接位点上游50bp的序列特征并鉴定motif。联用CPC和CNCI软件和比对Swiss-prot数据库的方法预测lncRNA,取三者的交集作为高可信度的lncRNA集合。进一步比较lncRNA和mRNA的转录本长度,外显子数量与长度,内含子长度,GC含...     

Ultrastructural study of spore wall morphogenesis inOphioglossum thermale kom. var.nipponicum (Miyabe et Kudo) nishida

Spore wall morphogenesis ofOphioglossum thermale var.nipponicum was examined by transmission electron microscopy. The spore wall of this species consists of three layers: endospore, exospore, and perispore. The spore wall development begins at the tetrad stage. At first, the outer undulating lamellar layer of the exospore (Lo) is formed on the spore plasma membrane in advance of the inner accumulating lamellar layer (Li) of the exospore. Next, the homogeneous layer of the exospore (H) is deposited on the outer lamellar layer. Both lamellar layers may be derived from spore cytoplasm; and the homogeneous layer, from the tapetum. Then the endospore (EN) is formed. It may be derived from spore cytoplasm. The membranous perispore (PE), derived from the tapetum, covers the exospore surface as the final layer. Though the ornamentation of this species differs distinctly from that ofO. vulgatum, the results mentioned above are fundamentally in accordance with the data obtained fromO. vulgatum (Lugardon, 1971). Therefore, the pattern of spore wall morphogenesis appears to be very stable in the genusOphioglossum.     

Influence of carbon dioxide on Nosema apis infection of honeybees (Apis mellifera)

Young workers of the honeybee Apis mellifera carnica were individually inoculated with Nosema apis spores subjected to carbon dioxide (CO(2)) treatment. The spores were kept in a CO(2) atmosphere for 30, 35 and 40 h. The course of the infection was evaluated on the basis of the survival rate of bee workers and the number of N. apis spores in their digestive tracts. CO(2) treatment of N. apis spores resulted in faster proliferation of the parasite as well as higher mortality among workers infected with spores kept in CO(2) for 30 and 35 h.     

Ultrastructural changes in major organelles during spermatial differentiation inBangia (Rhodophyta)

Summary The major organelles within the cells of maleBangia atropurpurea (Roth) C. Ag. filaments undergo a series of ultrastructural transformations during the production of spermatia. Initially, thylakoids within the large axial chloroplast develop a reticulate pattern commencing at the central pyrenoid region. Subsequent changes involve loss of lobes and diminution of volume through division; chloroplasts in final stages contain a few dilated, distorted thylakoids and many plastoglobuli. During differentiation the large nucleolus disappears from the nucleus and four masses of chromatin aggregate near the nuclear envelope. Furrows originating from the nuclear envelope form double membranes around each of the chromatin masses and most of the nucleoplasm is eliminated. Several types of fibrillar vesicles are formed during the process and large floridean starch reserves are utilized. Multilamellar bodies and microbody-like structures occur within the cells during certain phases of spermatiogenesis.     

Effect of L-cystine on macromolecular changes during spore and parasporal crystal formation inBacillus thuringiensis var.thuringiensis

High concentration of L-cystine (0.25%) when present in a glucose-mineral salt medium inhibited sporulation-specific events like protease production, calcium uptake and dipicolinic acid synthesis inBacillus thuringiensis var.thuringiensis. In addition, the enzymes of the Krebs cycle from aconitase onwards were completely inhibited by a high concentration of cystine. At a low concentration of cystine (0.05%), none of the above mentioned macromolecular changes were affected. Lipid synthesis monitored by [1,2¹⁴ C]-acetate incorporation into lipid as well as into whole cells was completely inhibited.     

Host-seeking behaviour of tracheal mites (Acari: Tarsonemidae) on honey bees (Hymenoptera: Apidae)

The behaviour of the endoparasitic tracheal mite, Acarapis woodi (Rennie) on honey bees (Apis mellifera L.) is a challenge to observe because of its small size. Through a microscope, we videotaped this mite's movement on young bees, dead bees and bees exposed to vegetable oil. Previous studies have shown that solid vegetable oil decreases mite infestations in a bee colony. We hypothesized that the oil alters mite behaviour to the detriment of the parasite, thus helping to safeguard the host. Habitat-seeking behaviour, identified as necessary for mites to locate a new host environment, was disrupted on both dead and oil-treated bees. Questing behaviour, which is associated with transfer between hosts, increased significantly on the dead and oily bees. The behaviours of mites were significantly different between all three treatments (x ²=494.96, p<0.001 on dead bees and x ²=851.11, p<0.001 on oily bees). Both questing and seeking behaviours were significantly different on each of the thoracic treatments (F _2,66=7.88, p<0.001 and F _2,66=21.28, p<0.001) and mite questing behaviour was not altered between males and females on live or oily bees (F _1,22=0.25, p<0.62), but habitat seeking was (F _1,22=7.42, p<0.012). The male questing and habitat-seeking behaviours were observed. We conclude that oil-treated bees gained protection from habitat-seeking mites because the normal behaviour of the mites seeking an oviposition site is interrupted.     

Fecundity,spore recruitment and size in Gelidium sesquipedale (Gelidiales,Rhodophyta)

Gelidium sesquipedale fecundity was quantified by counting tetrasporangial sori and cystocarps per meter squared and by estimating the number of spores contained inside them. These were obtained by regression on a size metric of reproductive structures. Tetrasporangial sori length and cystocarp thickness were the best estimators of spore number. To assess spore recruitment, 12 pottery tiles were fixed to the bottom, and the appearance of small fronds was monitored.No clear seasonal pattern of reproduction was found. Tetraspore production peaked in March 1990 with 10.4 × 10⁶ spores m^–2, whereas the carpospore peak was lower, 4.9 × 10⁵ spores m^–2 in July 1989. Recruitment followed tetraspore peaks. The probability of a G. sesquipedale tetraspore making the transition to a recruit was 4.7 × 10^–5. Frond length was significantly related to tetrasporangial sori number, while cystocarp number was only related to frond branching order. Minimum size for reproduction was 6.9 cm for gametophytes and 5.4 cm for tetrasporophytes; very rarely were cystocarpic fronds smaller than 9 cm, while tetrasporic fronds were often longer than 15 cm. Cystocarpic fronds were significantly shorter and had more branches than tetrasporic fronds.     

Transformation of Metarhizium anisopliae with benomyl resistance and green fluorescent protein genes provides a tag for genetically engineered strains

A plasmid, pBGFP, carrying green fluorescent protein (gfp) and benomyl-resistance genes was constructed and transformed into Metarhizium anisopliae. The transformants grew normally and GFP fluorescence was detected. No change was found in virulence for the transformants. Fluorescence was detected in hyphae from the haemolymph of the infected locust, and the benomyl-resistance was maintained. Results suggested that the two markers provided a useful tool for screening and monitoring the engineered strains even after infection.     

The Role of Cuticular Compounds in the Resistance of Honey Bees (Apis Mellifera) to Tracheal Mites (Acarapis Woodi)

This study examined the migration of tracheal mites (Acarapis woodi) into honey bees (Apis mellifera) from different colonies and the relative attraction of mites to hexane extracts from the external body surfaces of young bees. Relative resistance of bees from different colonies initially was assessed with a field bioassay that involved tagging newly emerged bees, pooling them in heavily mite-infested colonies, retrieving them 7 days later, and examining them for tracheal mite prevalence and abundance. For those colonies identified as most resistant and least resistant, cuticular chemicals were extracted in hexane from frozen, newly emerged worker bees. These extracts were presented to individual tracheal mites in pairwise fashion in a laboratory bioassay. The results demonstrated that mites prefer extracts of bees from some colonies more than others, however, no consistent differences were demonstrated. Our inability to predict mite responses to extracts based on our initial assessment of relative resistance indicates that other mechanisms of resistance influence mite success in colonizing new host bees.     

蜜蜂及熊蜂微孢子虫rRNA基因研究

核糖体RNA(rRNA)是一种理想的进化计时器,已广泛地应用于研究微生物的系统发育和进化关系。蜜蜂和熊蜂都被微孢子虫感染,关于蜜蜂和熊蜂微孢子虫rRNA基因研究主要有rRNA基因序列的测定分析、二级结构的研究和用rRNA基因检测诊断微孢子虫等。这些对蜜蜂和熊蜂微孢子虫系统发育的研究和微孢子虫的防治等具有重要的推动作用。     

中华蜜蜂幼虫肠道响应球囊菌胁迫的microRNA应答分析

【目的】蜜蜂球囊菌(Ascosphaera apis,简称球囊菌)是一种能够侵染中华蜜蜂(Apis cerana cerana,简称中蜂)幼虫的致死性真菌病原。微小RNA(microRNA,miRNA)可通过在转录后水平靶向抑制或降解mRNA而参与宿主与病原互作过程。本研究旨在对球囊菌胁迫的中蜂6日龄幼虫肠道的差异表达miRNA(DEmiRNA)及其靶基因进行深入分析,进而揭示DEmiRNA在中蜂响应球囊菌胁迫应答过程中的作用。【方法】利用Illumina MiSeq平台对正常及球囊菌胁迫的中蜂6日龄幼虫肠道(AcCK和AcT)进行测序,通过相关生物信息学软件预测DEmiRNA及其靶基因。通过Blast将靶基因注释到GO和KEGG数据库。利用Cytoscape软件构建DEmiRNA与其靶mRNA的调控网络。通过Stem-loop RT-PCR和qPCR验证测序数据的可靠性。【结果】本研究共预测出537个miRNA,其长度分布介于16–35 nt之间,且不同长度的miRNA首位碱基偏向性差异明显。通过Stem-loop RT-PCR证实了10个novel miRNA的表达。AcCK vs AcT比较组共有54个DEmiRNA,包含31个上调和23个下调miRNA,可分别靶向结合6170和8199个靶基因。GO分类结果显示上调和下调miRNA的靶基因分别涉及47和47个条目,富集基因数最多的皆为结合细胞进程和催化活性。KEGG代谢通路(pathway)富集分析结果表明上调和下调miRNA的靶基因分别富集在134和126条pathway,富集基因数最多的均为内吞作用和内质网中的蛋白质加工。调控网络分析结果表明,DEmiRNA及其靶mRNA形成十分复杂的调控关系;31个DEmiRNA可靶向结合51个与泛素介导的蛋白水解相关的mRNA,18个DEmiRNA可靶向结合14个与Jak-STAT信号通路相关的mRNA;miR-1277-x、miR-26-x、miR-27-y、miR-30-x、miR-6052-x等16个miRNA共同参与了上述两条免疫通路的调控。最后,随机挑选3个DEmiRNA进行qPCR验证,结果证明了测序数据的可靠性。【结论】本研究提供了中蜂幼虫肠道在球囊菌胁迫后期的miRNA的表达谱和差异表达信息,揭示了球囊菌与宿主之间在miRNA组学水平存在复杂的互作。miR-6052-x和miR-1277-x作为调控网络的核心可能通过影响细胞凋亡参与宿主的免疫防御,miR-26-x和miR-30-x可能通过调控Jak-STAT信号通路参与宿主的胁迫应答。本研究筛选出的关键DEmiRNA有望作为治疗白垩病的分子靶标。     

Ergonomics in stingless bees: changes in intranidal behavior after partial removal of storage pots and honey inMelipona favosa (Hym. Apidae,Meliponinae)

Summary The ergonomics of task allocation in stingless bees was examined in two laboratory colonies ofMelipona favosa containing individually marked workers. Performance of intranidal behavior patterns, brood cell production and the processing and storage of nectar, were studied during a control and a wax-deprived period. Experimental wax deprivation consisted of regular controlled removal of storage pots and their contents. Stored carbohydrate concentration and storage pot mass declined during the wax-deprived period. Behavior was measured by sampling for a complete ethogram at seven intranidal areas.In comparison withApis mellifera, M. favosa appeared to be more sensitive to stress, with a distinct reallocation of effort between tasks. The significant increase under stress of brood production and brood-rearing behavior patterns observed was previously also found inApis. Self-oriented behavior patterns declined under stressed circumstances.Involucrum construction declined in wax-deprivedMelipona colonies. Relatively low rates of walking behavior in general inMelipona suggest a low mean free distance between tasks. These results are of particular interest in relation to the basically different nest structure of stingless bees, e.g. very distinct areas for brood production and the processing and storage of food. This architecture of stingless bee nests appeared to influence strongly their specific responses to wax deprivation.     

Resting spore formation ofLeptocylindrus danicus (Bacillariophyceae) during short time-scale upwelling and its significance as predicted by a simple model

Resting spore formation during short time-scale upwelling and its significance were investigated in the field and by a simple theoretical model. Field observations of spore formation ofLeptocylindrus danicus were made off Izu Peninsula, Japan. A rapid increase in ratio of resting spore to vegetative cell numbers indicated thatL. danicus formed resting spores quickly as a response to nutrient depletion in the upwelled water, although only a very low number of resting spores was found in the upwelling. A simple model was constructed to investigate the possible advantages of spore formation during short time-scale upwelling. This showed that there is a critical time-scale for resting spore formation to be advantageous. The nutrient depletion period of the upwelling off Izu was shorter than the critical time-scale determined by the model. Rapid-sinking of resting spores may increase further the critical time-scale, unless spores return with upwelling water. For short time-scale upwelling, the vegetative cell may be better suited than the resting spore for enduring a short period of nutrient depletion. Contribution from Shimoda Marine Research Center, University of Tsukuba, No. 475.     

Selection on a haploid genotype for discrimination learning performance: Correlation between drone honey bees (Apis mellifera) and their worker progeny (Hymenoptera: Apidae)

Successful bidirectional selection for discriminative olfactory learning is reported for drone honey bees (Apis mellifera). Learning performance was evaluated using a discrimination conditioning procedure that required drones to discriminate between an appetitively reinforced odorant and one that was followed by punishment. Selective breeding produced high- and low-learning-performance lines of worker progeny that diverged from performance of workers whose fathers were selected at random. Furthermore, we show that levels of sucrose-induced sensitization are not correlated to learning performance. These results corroborate earlier findings and further demonstrate the power of selection on a haploid (drone) genotype. In addition, this study now shows that the demonstrated differences in learning performance cannot be completely accounted for by alteration of sucrose-induced sensitization. Thus, using this technique, it may be possible to select for associative conditioning without a pleiotropic increase in sensitization. The honey bee will be ideally suited to these types of correlation analyses in future studies.