The Effects of Nosema apis and Nosema ceranae Infection on Survival and Phenoloxidase Gene Expression in Galleria mellonella (Lepidoptera: Galleriidae) Compared to Apismellifera

The study aims to prove the possibility of colonization of N. apis and N. ceranae to the intestine of the greater wax moth, detect the differences of greater wax moth based on the presence of Nosema species and examine the effect of Nosema species on the phenoloxidase level of greater wax moth compared with honeybees. Each group was fed on the 1st day of the experiment with its appropriate diet containing 106 Nosema spores per insect. Each group was checked daily, and dead insects were counted. Furthermore, changes in the level of expression of the phenoloxidase-related gene after Nosema spp. treatment on the 6th, 9th and 12th days, which was detected by Q-PCR, and the mRNA level of phenoloxidase gene were measured in all experiment groups with the CFX Connect Real-Time PCR Detection System. This study shows that Apis mellifera L. has a 66.7% mortality rate in mixed Nosema infections, a 50% mortality rate in N. ceranae infection, a 40% mortality rate in N. apis infection, while there is no death in G. mellonella. A significant difference was found in the mixed Nosema infection group compared to the single Nosema infection groups by means of A. mellifera and G. mellonella (Duncan, p < 0.05). G. mellonella histopathology also shows that Nosema spores multiply in the epithelial cells of greater wax moth without causing any death. The increase in the mRNA level of Phenoloxidase gene in A. mellifera was detected (Kruskal–Wallis, p < 0.05), while the mRNA level of the Phenoloxidase gene did not change in G. mellonella (Kruskal–Wallis, p > 0.05). These findings prove that the Nosema species can colonize into the greater wax moth, which contributes to the dissemination of these Nosema species between beehives.

Effects of Agaricus bisporus Mushroom Extract on Honey Bees Infected with Nosema ceranae

Agaricus bisporus water crude extract was tested on honey bees for the first time. The first part of the cage experiment was set for selecting one concentration of the A. bisporus extract. Concentration of 200 µg/g was further tested in the second part of the experiment where bee survival and food consumption were monitored together with Nosema infection level and expression of five genes (abaecin, hymenoptaecin, defensin, apidaecin, and vitellogenin) that were evaluated in bees sampled on days 7 and 15. Survival rate of Nosema-infected bees was significantly greater in groups fed with A. bisporus-enriched syrup compared to those fed with a pure sucrose syrup. Besides, the anti-Nosema effect of A. bisporus extract was greatest when applied from the third day which coincides with the time of infection with N. ceranae. Daily food consumption did not differ between the groups indicating good acceptability and palatability of the extract. A. bisporus extract showed a stimulative effect on four out of five monitored genes. Both anti-Nosema and nutrigenomic effects of A. bisporus extract were observed when supplementation started at the moment of N. ceranae infection or preventively (before or simultaneously with the infection).

Association between the Microsatellite Ap243, AC117 and SV185 Polymorphisms and Nosema Disease in the Dark Forest Bee Apis mellifera mellifera

The microsporidian Nosema parasites, primarily Nosema ceranae, remain critical threats to the health of the honey bee Apis mellifera. One promising intervention approach is the breeding of Nosema-resistant honey bee colonies using molecular technologies, for example marker-assisted selection (MAS). For this, specific genetic markers used in bee selection should be developed. The objective of the paper is to search for associations between some microsatellite markers and Nosema disease in a dark forest bee Apis mellifera mellifera. For the dark forest bee, the most promising molecular genetic markers for determining resistance to nosemosis are microsatellite loci AC117, Ap243 and SV185, the alleles of which (“177”, “263” and “269”, respectively) were associated with a low level of Nosema infection. This article is the first associative study aimed at finding DNA loci of resistance to nosemosis in the dark forest bee. Nevertheless, microsatellite markers identified can be used to predict the risk of developing the Nosema disease.

Effects of Synthetic Acaricides and Nosema ceranae (Microsporidia: Nosematidae) on Molecules Associated with Chemical Communication and Recognition in Honey Bees

Acaricides and the gut parasite Nosema ceranae are commonly present in most productive hives. Those stressors could be affecting key semiochemicals, which act as homeostasis regulators in Apis mellifera colonies, such as cuticular hydrocarbons (CHC) involved in social recognition and ethyl oleate (EO) which plays a role as primer pheromone in honey bees. Here we test the effect of amitraz, coumaphos, tau-fluvalinate and flumethrin, commonly applied to treat varroosis, on honey bee survival time, rate of food consumption, CHC profiles and EO production on N. ceranae-infected and non-infected honey bees. Different sublethal concentrations of amitraz, coumaphos, tau-fluvalinate and flumethrin were administered chronically in a syrup-based diet. After treatment, purified hole-body extracts were analyzed by gas chromatography coupled to mass spectrometry. While N. ceranae infection was also shown to decrease EO production affecting survival rates, acaricides showed no significant effect on this pheromone. As for the CHC, we found no changes in relation to the health status or consumption of acaricides. This absence of alteration in EO or CHC as response to acaricides ingestion or in combination with N. ceranae, suggests that worker honey bees exposed to those highly ubiquitous drugs are hardly differentiated by nest-mates. Having determined a synergic effect on mortality in worker bees exposed to coumaphos and Nosema infection but also, alterations in EO production as a response to N. ceranae infection it is an interesting clue to deeper understand the effects of parasite-host-pesticide interaction on colony functioning.

Overt Infection with Chronic Bee Paralysis Virus (CBPV) in Two Honey Bee Colonies

Chronic Bee Paralysis Virus (CBPV), a widespread honey bee RNA virus, causes massive worker bee losses, mostly in strong colonies. Two different syndromes, with paralysis, ataxia and flight incapacity on one hand and black hairless individuals with shortened abdomens on the other, can affect a colony simultaneously. This case report presents two Apis mellifera carnica colonies with symptoms of paralysis and hairless black syndrome in 2019. Via RT-PCR, a highly positive result for CBPV was detected in both samples. Further problems, such as a Nosema infection and Varroa infestation, were present in these colonies. Therapy methods were applied to colony 1 comprising queen replacement, shook swarm method and Varroa control, whereas colony 2 was asphyxiated after queen loss and colony weakening. After therapy, colony 1 was wintered without symptoms. Beekeeping and sanitary measures can save a CBPV-infected colony, while further complications result in total colony loss.

Microscopic and Molecular Detection of Nosema ceranae in Honeybee Apis mellifera L. from Romania Status on pathogen worldwide distribution

Until now, in Romania, a country in southeastern Europe located on the Balkans Peninsula, information regarding the status on A. mellifera honey bee infection by Nosema spp. has not been published. The aims of the present study were to survey the occurrence and identification of Nosema spp. in A. mellifera honeybee colonies from 37 apiaries located in Arad, Caras-Severin and Timis Counties, Romania. Also, based on published literature an update on the distribution of N. ceranae infection among A. mellifera colonies worldwide was performed. Overall, a frequency of 55.1% (389/690) of Nosema infection was observed in the analyzed regions (ranging from 52% to 66%), by light microscopy. By PCR and DNA sequencing, N. ceranae was the only microsporidia identified. Nosema ceranae is clearly a novel, emergent pathogen of A.mellifera with potentially very serious effects on the individual and honeybee�s colonies in Romania. Data obtained provide new and important information on N. ceranae geographic prevalence and distribution, and on its impact at colony level and/or its role in colony losses. The present study intends to contribute to highlight the importance of implementing prevention, treatment and control measures of honeybee nosemosis, in Romania.