First detection of Paenibacillus larvae the causative agent of American Foulbrood in a Ugandan honeybee colony

Paenibacillus larvae is a highly contagious and often lethal widely distributed pathogen of honeybees, Apis mellifera but has not been reported in eastern Africa to date. We investigated the presence of P. larvae in the eastern and western highland agro-ecological zones of Uganda by collecting brood and honey samples from 67 honeybee colonies in two sampling occasions and cultivated them for P. larvae. Also, 8 honeys imported and locally retailed in Uganda were sampled and cultivated for P. larvae. Our aim was to establish the presence and distribution of P. larvae in honeybee populations in the two highland agro-ecological zones of Uganda and to determine if honeys that were locally retailed contained this lethal pathogen. One honeybee colony without clinical symptoms for P. larvae in an apiary located in a protected area of the western highlands of Uganda was found positive for P. larvae. The strain of this P. larvae was genotyped and found to be ERIC I. In order to compare its virulence with P. larvae reference strains, in vitro infection experiments were conducted with carniolan honeybee larvae from the research laboratory at Ghent University, Belgium. The results show that the virulence of the P. larvae strain found in Uganda was at least equally high. The epidemiological implication of the presence of P. larvae in a protected area is discussed

Nosema neumanni n. sp. (Microsporidia, Nosematidae), a new microsporidian parasite of honeybees, Apis mellifera in Uganda

The microsporidium Nosema neumanni n. sp., a new parasite of the honeybee Apis mellifera is described based on its ultra structural and molecular characteristics. Structures resembling microsporidian spores were found by microscopic examination of honeybees from Uganda. Molecular confirmation failed when PCR primers specific for Nosema apis and Nosema ceranae were used, but was successful with primers covering the whole family of Nosematidae. We performed transmission electron microscopy and found typical microsporidian spores which were smaller (length: 2.36 +/- 0.14 m and width: 1.78 +/- 0.06 p.m; n = 6) and had fewer polar filament coils (10-12) when compared to those of known species infecting honeybees. The entire 16S SS(J rRNA region was amplified, cloned and sequenced and was found to be unique with the highest resemblance (97% identity) to N. apis, The incidence of N. neumanni n. sp. in Ugandan honeybees was found to be much higher than of the two other Nosema species

Comparison between Apicystis cryptica sp. n. and Apicystis bombi (Arthrogregarida, Apicomplexa): Gregarine parasites that cause fat body hypertrophism in bees

The molecular divergence, morphology and pathology of a cryptic gregarine that is related to the bee parasite Apicystis bombi Lipa and Triggiani, 1996 is described. The 18S ribosomal DNA gene sequence of the new gregarine was equally dissimilar to that of A. bombi and the closest related genus Mattesia Naville, 1930, although phylogenetic analysis supported a closer relation to A. bombi. Pronounced divergence with A. bombi was found in the ITS1 sequence (69.6% similarity) and seven protein-coding genes (nucleotide 78.05% and protein 90.2% similarity). The new gregarine was isolated from a Bombus pascuorum Scopoli, 1763 female and caused heavy hypertrophism of the fat body tissue in its host. In addition, infected cells of the hypopharyng ealgland tissue, an important excretory organ of the host, were observed. Mature oocysts were navicular in shape and contained four sporozoites, similar to A. bombi oocysts. Given these characteristics, we proposed the name Apicystis cryptica sp. n. Detections so far indicated that distribution and host species occupation of Apicystis spp. overlap at least in Europe, and that historical detections could not discriminate between them. Specific molecular assays were developed that can be implemented in future pathogen screens that aim to discriminate Apicystis spp. in bees. (C) 2020 The Authors. Published by Elsevier GmbH

Bee pathogens found in Bombus atratus from Colombia : a case study

Bombus atratus bumblebees from Colombia that were caught in the wild and from breeding programs were screened for a broad set of bee pathogens. We discovered for the first time Lake Sinai Virus and confirmed the infection by other common viruses. The prevalence of Apicystis bombi, Crithidia bombi and Nosema ceranae was remarkably high. According to other studies the former two could have been co-introduced in South America with exotic bumble bees as Bombus terrestris or Bombus ruderatus. Given the fact that none of these species occur in Colombia, our data puts a new light on the spread of these pathogens over the South American continent

Heritability estimates of the novel trait 'suppressed in ovo virus infection' in honey bees (Apis mellifera)

Honey bees are under pressure due to abnormal high colony death rates, especially during the winter. The infestation by the Varroa destructor mite and the viruses that this ectoparasite transmits are generally considered as the bees' most important biological threats. Almost all efforts to remedy this dual infection have so far focused on the control of the Varroa mite alone and not on the viruses it transmits. In the present study, the sanitary control of breeding queens was conducted on eggs taken from drone brood for 4 consecutive years (2015-2018). The screening was performed on the sideline of an ongoing breeding program, which allowed us to estimate the heritabilities of the virus status of the eggs. We used the term 'suppressed in ovo virus infection' (SOV) for this novel trait and found moderate heritabilities for the presence of several viruses simultaneously and for the presence of single viral species. Colonies that expressed the SOV trait seemed to be more resilient to virus infections as a whole with fewer and less severe Deformed wing virus infections in most developmental stages, especially in the male caste. The implementation of this novel trait into breeding programs is recommended

Reference Gene Selection for Insect Expression Studies Using Quantitative Real-Time PCR: The Head of the Honeybee, Apis mellifera, After a Bacterial Challenge

In this study an important and often neglected aspect of gene expression studies in insects, the validation of appropriate reference genes with stable expression levels between sample groups, is addressed. Although in this paper the reference gene selection for the honeybee, Apis mellifera L. (Hymenoptera: Apidae) head was tested in the context of bacterial challenge with Escherichia coli, this work can serve as a resource to help select and screen insect reference genes for gene expression studies in any tissue and under any experimental manipulation. Since it is recommended to use multiple reference genes for accurate normalization, we analyzed the expression of eleven candidate reference genes in the honeybee head, for their potential use in the analysis of differential gene expression following bacterial challenge. Three software programs, BestKeeper, Normfinder and geNorm, were used to assess candidate reference genes. GeNorm recommended the use of four reference genes. Both geNorm and Normfinder identified the genes GAPDH, RPS18, actin and RPL13a as the most stable ones, only differing in their ranking order. BestKeeper identified RPS18 as being the reference gene with the least overall variation, but also actin and GAPDH were found to be the second and third most stable expressed gene. By a combination of three software programs the genes actin, RPS18 and GAPDH were found suitable reference genes in the honeybee head in the context of bacterial infection

Extending the honey bee venome with the antimicrobial peptide apidaecin and a protein resembling wasp antigen 5

Honey bee venom is a complex mixture of toxic proteins and peptides. In the present study we tried to extend our knowledge of the venom composition using two different approaches. First, worker venom was analysed by liquid chromatography-mass spectrometry and this revealed the antimicrobial peptide apidaecin for the first time in such samples. Its expression in the venom gland was confirmed by reverse transcription PCR and by a peptidomic analysis of the venom apparatus tissue. Second, genome mining revealed a list of proteins with resemblance to known insect allergens or venom toxins, one of which showed homology to proteins of the antigen 5 (Ag5)/Sol i 3 cluster. It was demonstrated that the honey bee Ag5-like gene is expressed by venom gland tissue of winter bees but not of summer bees. Besides this seasonal variation, it shows an interesting spatial expression pattern with additional production in the hypopharyngeal glands, the brains and the midgut. Finally, our immunoblot study revealed that both synthetic apidaecin and the Ag5-like recombinant from bacteria evoke no humoral activity in beekeepers. Also, no IgG4-based cross-reactivity was detected between the honey bee Ag5-like protein and its yellow jacket paralogue Ves v 5

Virus Prevalence in Egg Samples Collected from Naturally Selected and Traditionally Managed Honey Bee Colonies across Europe

Monitoring virus infections can be an important selection tool in honey bee breeding. A recent study pointed towards an association between the virus-free status of eggs and an increased virus resistance to deformed wing virus (DWV) at the colony level. In this study, eggs from both naturally surviving and traditionally managed colonies from across Europe were screened for the prevalence of different viruses. Screenings were performed using the phenotyping protocol of the 'suppressed in ovo virus infection' trait but with qPCR instead of end-point PCR and a primer set that covers all DWV genotypes. Of the 213 screened samples, 109 were infected with DWV, 54 were infected with black queen cell virus (BQCV), 3 were infected with the sacbrood virus, and 2 were infected with the acute bee paralyses virus. It was demonstrated that incidences of the vertical transmission of DWV were more frequent in naturally surviving than in traditionally managed colonies, although the virus loads in the eggs remained the same. When comparing virus infections with queen age, older queens showed significantly lower infection loads of DWV in both traditionally managed and naturally surviving colonies, as well as reduced DWV infection frequencies in traditionally managed colonies. We determined that the detection frequencies of DWV and BQCV in honey bee eggs were lower in samples obtained in the spring than in those collected in the summer, indicating that vertical transmission may be lower in spring. Together, these patterns in vertical transmission show that honey bee queens have the potential to reduce the degree of vertical transmission over time