Impact of Nosema ceranae invasion on sucrose solution consumption, midgut epithelial cell structure, and lifespan of Apis cerana cerana workers

Nosema ceranae is an intracellular fungal parasite for honeybees, leading to chronic disease named bee nosemosis with worldwide distribution. Asian honeybee (Apis cerana) is the original host for N. ceranae, but the impact of N. ceranae infection on A. cerana physiology is largely unknown. In this current work, workers of Apis cerana cerana, a subspecies of Asian honeybee, were artificially inoculated with N. ceranae spores and reared under lab conditions, followed by detection of fungal spore load as well as host sucrose solution consumption, midgut epithelial cell structure, and lifespan. The result of spore counting suggested that the spore load in the host midgut decreased significantly during 1 dpi-2 dpi, whereas that displayed an elevated trend among 2 dpi-13 dpi. The sucrose solution consumption of workers in N. ceranae-inoculated groups among 1 dpi-20 dpi was always higher than that of workers in un-inoculated groups; additionally, the difference of sucrose solution consumption between these two groups at 4 dpi, 5 dpi, and 13 dpi was of significance. Based on microscopic observation of paraffin sections, darkly stained parasites were clearly detected in the midgut epithelial cells of N. ceranae-inoculated workers at 7 dpi-10 dpi, whereas no parasite was observed in those of un-inoculated workers. In addition, the boundaries of un-inoculated host epithelial cells were intact and the darkly stained nucleus were clear, while the boundaries of midgut epithelial cells of N. ceranae-inoculated workers were blurred, the nucleus were almost disappeared, and the nucleic acid substances were diffused. Moreover, the survival rates of workers in both N. ceranae-inoculated groups and un-inoculated groups at 1 dpi-5 dpi were pretty high and then started to decrease at 5 dpi; the survival rate of workers in N. ceranae-inoculated groups was always lower than that in un-inoculated groups, with significant difference between these two groups during 11 dpi-20 dpi. These results together indicate that the quantity of fungal spores continuously elevated with the microsporidian multiplication, causing energetic stress for workers and host cell structure damage, which further negatively affected the host lifespan. Our findings offer a solid basis not only for exploring the molecular mechanism underlying N. ceranae infection but also for investigating the interaction between N. ceranae and eastern honeybee.

Reproductive Potential Impacts Body Maintenance Parameters and Global DNA Methylation in Honeybee Workers (Apis mellifera L.)

The widely accepted hypothesis in life history evolution about the trade-off between fecundity and longevity is not confirmed by long-living and highly fecund queens in eusocial insects. The fact that the queens and facultatively sterile workers usually arise from genetically identical eggs but differ in DNA methylation makes them a good model for studies on senescence, eusocial evolution, and epigenetics. Therefore, honeybees seem to be especially useful here because of long living rebel-workers (RW) with high reproductive potential recently described. Longevity, ovariole number, nosema tolerance, and global DNA methylation have been assayed in normal workers (NW) versus RW in hives and cages. RW always lived longer than NW and unexpectedly extended longevity of NW when they were together, similarly as the presence of a queen did. RW lived longer despite the fact that they had higher Nosema spore load; surprisingly they became infected more easily but tolerated the infection better. Global DNA methylation increased with age, being lower in RW than in NW. Therefore, RW are queen-like considering global DNA methylation and the link between fecundity, longevity, and body maintenance. Presented features of RW expands possibilities of the use of honeybees as a model for studies on senescence, nosemosis, eusocial evolution, and epigenetics.

Tilletia species on winter wheat grain

Goal. To determine the species composition of the complex of pathogens of bunt diseases of the genus Tilletia on winter wheat grain. Methods. The research was conducted during 2017—2020. 315 samples of winter wheat grain from different soil and climatic zones were analyzed. Seed contamination was determined by washing the grains and centrifuging the spore suspension, followed by counting their number in the Goryaev chamber. Identification of the pathogen was performed in the laboratory by microscopic examination, taking into account the morphological features of teliospores. Results. In 2017 and 2018, the presence of spores of fungi of the genus Tilletia was detected in 23.5 and 28.6% of the analyzed wheat grain samples, respectively. The number of spores per grain varied between 11—388.9 and 2.8—214.8 and averaged 72.5 and 33.7 correspondingly. The largest number of infected samples was observed in 2019 — 47.4%. In the same period, the highest level of seed contamination was observed — up to 1089 spores per grain, in average — 124.6 spores per grain. The lowest level of grain contamination was detected in 2020 — 6.3% of samples with an average of 3.7 spores per grain. Mostly spores of bunt pathogens were found in grain samples from Ternopil, Kyiv, Chernihiv, Lviv, Vinnytsia regions. The following species have been identified: Tilletia caries, T. controversa, T. laevis. Pathogen T. caries was detected annually. It dominated in 2020 and ranked second in detection rate in 2017—2019. T. controversa was found on grain in 2017—2019 and prevailed among other species. Its part in the complex of pathogens varied from 77.1 to 87.5%. In 2018, T. laevis was also detected. Conclusions. In most samples, spores of fungi of the genus Tilletia were not detected. On average, according to the years of research, grain contamination by bunt fungi were detected in 6.3—47.4% of samples with an average spore load of 3.7—124.6 of grain. The highest number of infected samples and the level of contamination were observed in 2019, the lowest — in 2020. The complex of pathogens is represented by three species: T. caries, T. laevis, T. controversa. T. controversa dominated in 2017—2019 and T. caries — in 2020. T. laevis occurred sporadically.

COMPARISON OF MALASSEZIA SPP. PROPORTIONS IN INFLAMMATORY AND NON-INFLAMMATORY FACIAL ACNE VULGARIS LESIONS

Objective: The skin microbiome plays a role in the pathogenesis of acne vulgaris (AV). Among other species, Malassezia spp. can be found in the pilosebaceous follicle. However, its role on the pathogenesis of AV has not yet been described. The objective of this study was to identify the presence and the distribution of Malassezia spp. in facial AV lesions and also to compare the distribution of Malassezia spp. between inflammatory and non-inflammatory lesions. Methods: One hundred and twenty subjects were allocated into two groups: inflammatory lesions and non-inflammatory lesions. Samples were taken from hair follicles and examined by microscopy using 20% potassium hydroxide and Parker ink and cultured for species identification. Results: There was no difference in the spore load between the two groups (p=0.142). Malassezia spp. were isolated from 64.2% of subjects and consisted of M. dermatis (43%), M. sympodialis (18%), M. slooffiae (16%), M. japonica (5%), M. furfur (5%), M. pachydermatis (3%), and M. restricta (1%). There was a higher Malassezia spp. proportion in non-inflammatory lesions (p=0.019). The predominant species in inflammatory AV lesions was M. dermatis (45,8%), followed by M. sympodialis (17.1%), and M. slooffiae (11.4%). Conclusion: Malassezia spp. were found in facial acne lesions. M. dermatis was the predominant species found in facial AV, followed by M. sympodialis, and M. slooffiae. A higher proportion of Malassezia spp. was found in non-inflammatory lesions.

Propolis Consumption Reduces Nosema ceranae Infection of European Honey Bees (Apis mellifera)

Nosema ceranae is a widespread obligate intracellular parasite of the ventriculus of many species of honey bee (Apis), including the Western honey bee Apis mellifera, in which it may lead to colony death. It can be controlled in A. mellifera by feeding the antibiotic fumagillin to a colony, though this product is toxic to humans and its use has now been banned in many countries, so in beekeeping, there exists a need for alternative and safe products effective against N. ceranae. Honeybees produce propolis from resinous substances collected from plants and use it to protect their nest from parasites and pathogens; propolis is thought to decrease the microbial load of the hive. We hypothesized that propolis might also reduce N. ceranae infection of individual bees and that they might consume propolis as a form of self-medication. To test these hypotheses, we evaluated the effects of an ethanolic extract of propolis administered orally on the longevity and spore load of experimentally N. ceranae-infected worker bees and also tested whether infected bees were more attracted to, and consumed a greater proportion of, a diet containing propolis in comparison to uninfected bees. Propolis extracts and ethanol (solvent control) increased the lifespan of N. ceranae-infected bees, but only propolis extract significantly reduced spore load. Our propolis extract primarily contained derivatives of caffeic acid, ferulic acid, ellagic acid and quercetin. Choice, scan sampling and food consumption tests did not reveal any preference of N. ceranae-infected bees for commercial candy containing propolis. Our research supports the hypothesis that propolis represents an effective and safe product to control N. ceranae but worker bees seem not to use it to self-medicate when infected with this pathogen.

Dynamics of Accumulation of Aerosols and Microscopic Fungi Spores in the Air of a Hermetic Inhabited Object during a Four-Month Isolation Experiment SIRIUS-19

A four-month experiment which reproduced the main temporal characteristics of the real space flight to the Moon in a medico-technical complex has been carried out. The content of aerosols in the indoor air of the hermetic inhabited object was continuously monitored using a Handheld 3016 laser aerosol counter (Lighthouse WS Co, United States). The number of particles was counted automatically, and the number of viable propagules and the species composition of the fungal population were determined by seeding. The results obtained by these two methods were compared, the regularities in dynamics of neutral particles and micromycete spores, as well as their relationship with the crew activity were revealed, and a possibility to use a particle counter on a spacecraft as an indicator of the microbiological situation was analyzed. A general tendency of an increase in the content of fungal spores in the air of the examined premises was observed from 2--5 at the beginning of the experiment to 287 ± 36 CFU/m3 closer to its end; however, this value did not exceed the sanitary standard recommended by WHO. Species potentially harmful for human health (Aspergillus fumigatus, A. niger and A. sydowii) were detected, but their spore load was extremely low. indoor air, spaceships, microbiological contamination, aerosol particle counter The work was supported by the National Research Center "Kurchatov Institute" (decree no. 1807 from 14.08.2019) and by the Moscow State University (grant for Leading Scientific Schools «Depository of the Living Systems» within the framework of the MSU Development Program).

Measurements of Aerial Spore Load by qPCR Facilitates Lettuce Downy Mildew Risk Advisement

The lettuce downy mildew pathogen, Bremia lactucae, is an obligate oomycete that causes extensive produce losses. Initial chlorotic symptoms that severely reduce the market value of the produce are followed by the appearance of white, downy sporulation on the abaxial side of the leaves. These spores become airborne and disseminate the pathogen. Controlling lettuce downy mildew has relied on repeated fungicide applications to prevent outbreaks. However, in addition to direct economic costs, heterogeneity and rapid adaptation of this pathogen to repeatedly applied fungicides has led to the development of fungicide-insensitivity in the pathogen. We deployed a quantitative PCR assay-based detection method using a species-specific DNA target for B. lactucae coupled with a spore trap system to measure airborne B. lactucae spore loads within three commercial fields that each contained experimental plots, designated EXP1 to EXP3. Based upon these measurements, when the spore load in the air reached a critical level (8.548 sporangia per m3 air), we advised whether or not to apply fungicides on a weekly basis within EXP1 to EXP3. This approach saved three sprays in EXP1, and one spray each in EXP2 and EXP3 without a significant increase in disease incidence. The reduction in fungicide applications to manage downy mildew can decrease lettuce production costs while slowing the development of fungicide resistance in B. lactucae by eliminating unnecessary fungicide applications.

Encephalitozoon intestinalis: A new target for auranofin in a mice model

Despite the fact that many approaches have been developed over years to find efficient and well-tolerated therapeutic regimens for microsporidiosis, the effectiveness of current drugs remains doubtful, and effective drugs against specific targets are still scarce. The present study is the first that was designed to evaluate the potency of auranofin, an anti-rheumatoid FDA approved drug, against intestinal Encephalitozoon intestinalis. Evaluation of the drug was achieved through counting of fecal and intestinal spores, studying the intestinal histopathological changes, measuring of intestinal hydrogen peroxide level, and post therapy follow-up of mice for 2 weeks for detection of relapse. Results showed that auranofin has promising anti-microsporidia potential. It showed a promising efficacy in mice experimentally infected with E. intestinalis. It has revealed an obvious reduction in fecal spore shedding and intestinal tissue spore load, amelioration of intestinal tissue pathological changes, and improvement of the local inflammatory infiltration without significant changes in hydrogen peroxide level. Interestingly, auranofin prevented the relapse of infection. Thus, considering the results of the present work, auranofin could be considered a therapeutic alternative for the gold standard drug ‘albendazole’ against the intestinal E. intestinalis infection especially in relapsing cases.