Population genetic diversity and dynamics of the honey bee brood pathogen Melissococcus plutonius in a region with high prevalence.

European foulbrood (EFB) is a honey bee brood disease caused by the bacterium Melissococcus plutonius. Large-scale EFB outbreaks have been reported in several countries in recent decades, which entail costly sanitation measures of affected apiaries to restrict the spread of this contagious pathogen. To mitigate its impact, a better understanding of the population dynamics of the etiological agent is required. We here used multi-locus sequence typing (MLST) to infer the genetic diversity and geographical distribution of 160 M. plutonius isolates collected from EFB symptomatic honey bee colonies seven years apart. Isolates belonged to three clonal complexes (CCs) known worldwide and to 12 sequence types (STs), of which five were novel. Phylogenetic and clustering analyses showed that some of these novel sequence types have likely evolved locally during a period of outbreak, but most disappeared again. We further screened the isolates for melissotoxin A (mtxA), a putative virulence gene. The prevalence of STs in which mtxA was frequent increased over time, suggesting that this gene promotes spread. Despite the increased frequency of this gene in the population, the total number of cases decreased, which could be due to stricter control measures implemented before the second sampling period. Our results provide a better understanding of M. plutonius population dynamics and help identify knowledge gaps that limit efficient control of this emerging disease.

In the battle of the disease: a transcriptomic analysis of European foulbrood-diseased larvae of the Western honey bee (Apis mellifera).

BACKGROUND: European foulbrood is a significant bacterial brood disease of Apis sp. and can cause severe and devastating damages in beekeeping operations. Nevertheless, the epidemiology of its causative agent Melissococcus plutonius has been begun to uncover but the underlying mechanisms of infection and cause of disease still is not well understood. Here, we sought to provide insight into the infection mechanism of EFB employing RNAseq in in vitro reared Apis mellifera larvae of two developmental stages to trace transcriptional changes in the course of the disease, including Paenibacillus alvei secondary infected individuals. RESULTS: In consideration of the progressing development of the larva, we show that infected individuals incur a shift in metabolic and structural protein-encoding genes, which are involved in metabolism of crucial compounds including all branches of macronutrient metabolism, transport protein genes and most strikingly chitin and cuticle associated genes. These changes underpin the frequently observed developmental retardation in EFB disease. Further, sets of expressed genes markedly differ in different stages of infection with almost no overlap. In an earlier stage of infection, a group of regulators of the melanization response cascade and complement component-like genes, predominantly C-type lectin genes, are up-regulated while a differential expression of immune effector genes is completely missing. In contrast, late-stage infected larvae up-regulated the expression of antimicrobial peptides, lysozymes and prominent bacteria-binding haemocyte receptor genes compared to controls. While we clearly show a significant effect of infection on expressed genes, these changes may partly result from a shift in expression timing due to developmental alterations of infection. A secondary infection with P. alvei elicits a specific response with most of the M. plutonius associated differential immune effector gene expression missing and several immune pathway genes even down-regulated. CONCLUSION: We conclude that with progressing infection diseased individuals undergo a systemic response with a change of metabolism and their activated immune defence repertoire. Moreover, larvae are capable of adjusting their response to a secondary invasion in late stage infections.

Field evaluation of Varroa-resistance traits in surviving Apis mellifera colonies in Argentina.

Varroa destructor is one of the most important sanitary threats for the beekeeping industry and so far disease control is based mainly on chemical treatment. However, a long-term solution may arise from studying natural surviving colonies of Apis mellifera. We compared the Varroa infestation rate in six commercial colonies that received annual treatment against mites and six non-treated colonies that survived in absence of any treatment for the last 6 years. In addition, we evaluated two potential mechanisms that might be involved in colony survival: hygienic (HYG) and Varroa-sensitive hygiene behavior (VSH) by means of pin-killed and mite artificial infestation, respectively. HYG and VSH were negatively correlated with mite infestation independently of the colony group (treated or non-treated). Furthermore, colonies expressing high levels of pupae removal (≥ 80%) showed higher %HYG and lower mite infestation compared to colonies showing low pupae removal (< 80%). The analysis of reproductive status of mites from the non-removed infested cells evidenced that more infertile mites are found in colonies with more than 80% of pupae removal. To study non-treated colonies that survive for several years, it is a suitable approach for identifying the underlying mechanisms related to Varroa-resistance.

European Foulbrood in stingless bees (Apidae: Meliponini) in Brazil: Old disease, renewed threat.

Stingless bees (Apidae: Meliponini) are a group of bees with vestigial stings showing a high level of social organization. They are important pollinators in tropical and subtropical regions, and, in the last decades, stingless beekeeping has increased rapidly in Brazil. Bee-collected pollen and honey of Apis mellifera can be an important source of disease when used as supplements to feed stingless bee colonies, a common and increasing practice adopted by stingless beekeepers. Here, we aimed to investigate the presence of pathogens commonly found in honey bees in diseased colonies of Melipona species in Espírito Santo and São Paulo States, Southeast Brazil. We detected, for the first time, the bacterium Melissococcus plutonius and symptoms of European foulbrood in Melipona spp., associated with brood death and colony losses in some cases. In addition, we tested for the presence of the bacterium Paenibacillus larvae and the fungus Aschosphaera apis, as well as the six more common honey bee viruses in Brazil (BQCV, ABPV, DWV, KBV, IAPV, CBPV) and the microsporidia Nosema apis and Nosema ceranae. However, only one sample of brood was infected with N. ceranae and all other pathogens, with the exception of Melissococcus plutonius, were absent in the analyzed brood. Lastly, we looked for toxic pollen in all food fed to diseased colonies, but none was present.

Virulence of Melissococcus plutonius and secondary invaders associated with European foulbrood disease of the honey bee.

European foulbrood is a globally distributed brood disease affecting honey bees. It may lead to lethal infections of larvae and, in severe cases, even to colony collapse. Lately, a profound genetic and phenotypic diversity was documented for the causative agent Melissococcus plutonius. However, experimental work on the impact of diverse M. plutonius strains on hosts with different genetic background is completely lacking and the role of secondary invaders is poorly understood. Here, we address these issues and elucidate the impact and interaction of both host and pathogen on one another. Moreover, we try to unravel the role of secondary bacterial invasions in foulbrood-diseased larvae. We employed in vitro infections with honey bee larvae from queens with different genetic background and three different M. plutonius strains. Larvae infection experiments showed host-dependent survival dynamics although M. plutonius strain 49.3 consistently had the highest virulence. This pattern was also reflected in significantly reduced weights of 49.3 strain-infected larvae compared to the other treatments. No difference was found in groups additionally inoculated with a secondary invader (Enterococcus faecalis or Paenibacillus alvei) neither in terms of larval survival nor weight. These results suggest that host background contributes markedly to the course of the disease but virulence is mainly dependent on pathogen genotype. Secondary invaders following a M. plutonius infection do not increase disease lethality and therefore may just be a colonization of weakened and immunodeficient, or dead larvae.

Comparative Genomics and Description of Putative Virulence Factors of Melissococcus plutonius, the Causative Agent of European Foulbrood Disease in Honey Bees.

In Europe, approximately 84% of cultivated crop species depend on insect pollinators, mainly bees. Apis mellifera (the Western honey bee) is the most important commercial pollinator worldwide. The Gram-positive bacterium Melissococcus plutonius is the causative agent of European foulbrood (EFB), a global honey bee brood disease. In order to detect putative virulence factors, we sequenced and analyzed the genomes of 14 M. plutonius strains, including two reference isolates. The isolates do not show a high diversity in genome size or number of predicted protein-encoding genes, ranging from 2.021 to 2.101 Mbp and 1589 to 1686, respectively. Comparative genomics detected genes that might play a role in EFB pathogenesis and ultimately in the death of the honey bee larvae. These include bacteriocins, bacteria cell surface- and host cell adhesion-associated proteins, an enterococcal polysaccharide antigen, an epsilon toxin, proteolytic enzymes, and capsule-associated proteins. In vivo expression of three putative virulence factors (endo-alpha-N-acetylgalactosaminidase, enhancin and epsilon toxin) was verified using naturally infected larvae. With our strain collection, we show for the first time that genomic differences exist between non-virulent and virulent typical strains, as well as a highly virulent atypical strain, that may contribute to the virulence of M. plutonius. Finally, we also detected a high number of conserved pseudogenes (75 to 156) per genome, which indicates genomic reduction during evolutionary host adaptation.

The Curious Case of Achromobacter eurydice, a Gram-Variable Pleomorphic Bacterium Associated with European Foulbrood Disease in Honeybees.

Honeybees are prone to parasite and pathogen infestations/infections due to their social colony life. Bacterial pathogens in particular lead to destructive infections of the brood. European foulbrood is caused by the bacterium Melissococcus plutonius in combination with several other Gram-positive bacteria (Achromobacter eurydice, Bacillus pumilus, Brevibacillus laterosporus, Enterococcus faecalis, Paenibacillus alvei, Paenibacillus dendritiformis) involved as secondary invaders following the initial infection. More than a century ago, A. eurydice was discovered to be associated with European foulbrood and morphologically and biochemically characterized. However, since the 1950s-1960s, only a few studies are known covering the biological relevance of this bacterium. Here, we review the biology, ecology, morphology, and biochemistry and discuss the still unclear systematic classification of A. eurydice.

First confirmed report of a bacterial brood disease in stingless bees.

Susceptibility to brood pathogens in eusocial stingless bees (Meliponini), alternative pollinators to honey bees, is unknown. Brood losses in managed colonies of the Australian stingless bee, Tetragonula carbonaria, were studied over 20months. We isolated a disease-causing bacterium, Lysinibacillus sphaericus (Firmicutes, Bacillaceae), from worker and queen larvae, brood cell provisions and honey stores. Pathogenicity experiments confirmed this bacterium as the causal organism. It took 22days from infection to first appearance of brood disease symptoms. This is the first confirmed record of a brood pathogen in stingless bees.