Influence of Age of Infection on the Gut Microbiota in Worker Honey Bees (Apis mellifera iberiensis) Experimentally Infected with Nosema ceranae.

The gut microbiota of honey bees has received increasing interest in the past decades due to its crucial role in their health, and can be disrupted by pathogen infection. Nosema ceranae is an intracellular parasite that affects the epithelial cells of the midgut, altering gut homeostasis and representing a major threat to honey bees. Previous studies indicated that younger worker bees are more susceptible to experimental infection by this parasite, although the impact of infection and of age on the gut bacterial communities remains unclear. To address this, honey bees were experimentally infected with a consistent number of N. ceranae spores at various ages post-emergence (p.e.) and the gut bacteria 7 days post-infection (p.i.) were analysed using real-time quantitative PCR, with the results compared to non-infected controls. Infected bees had a significantly higher proportion and load of Gilliamella apicola. In respect to the age of infection, the bees infected just after emergence had elevated loads of G. apicola, Bifidobacterium asteroides, Bombilactobacillus spp., Lactobacillus spp., Bartonella apis, and Bombella apis. Moreover, the G. apicola load was higher in bees infected at nearly all ages, whereas older non-infected bees had higher loads of Bifidobacterium asteroides, Bombilactobacillus spp., Lactobacillus spp., Ba. apis, and Bo apis. These findings suggest that N. ceranae infection and, in particular, the age of bees at infection modulate the gut bacterial community, with G. apicola being the most severely affected species.

Age and Method of Inoculation Influence the Infection of Worker Honey Bees (Apis mellifera) by Nosema ceranae.

The microsporidian parasite Nosema ceranae is a highly prevalent, global honey bee pathogen. Apis mellifera is considered to be a relatively recent host for this microsporidia, which raises questions as to how it affects its host's physiology, behavior and longevity, both at the individual and colony level. As such, honey bees were inoculated with fresh purified spores of this pathogen, both individually (Group A) or collectively (Group B) and they were studied from 0 to 15 days post-emergence (p.e.) to evaluate the effect of bee age and the method of inoculation at 7 days post-infection. The level of infection was analyzed individually by qPCR by measuring the relative amount of the N. ceranae polar tubule protein 3 (PTP3) gene. The results show that the bee's age and the method of infection directly influence parasite load, and thus, early disease development. Significant differences were found regarding bee age at the time of infection, whereby the youngest bees (new-born and 1 day p.e.) developed the highest parasite load, with this load decreasing dramatically in bees infected at 2 days p.e. before increasing again in bees infected at 3-4 days p.e. The parasite load in bees infected when older than 4 days p.e. diminished as they aged. When the age cohort data was pooled and grouped according to the method of infection, a significantly higher mean concentration and lower variation in N. ceranae infection was evident in Group A, indicating greater variation in experimental infection when spores were administered collectively to bees through their food. In summary, these data indicate that both biological and experimental factors should be taken into consideration when comparing data published in the literature.

The levels of natural Nosema spp. infection in Apis mellifera iberiensis brood stages.

Nosema ceranae is the most prevalent endoparasite of Apis mellifera iberiensis and it is a major health problem for bees worldwide. The infective capacity of N. ceranae has been demonstrated experimentally in honey bee brood, however no data are available about its prevalence in brood under natural conditions. Thus, brood combs from 10 different hives were analyzed over two consecutive years, taking samples before and after winter. A total of 1433 larvae/pupae were analyzed individually and N. ceranae (3.53%) was the microsporidian most frequently detected, as opposed to Nosema apis (0.42%) which was more frequently detected in conjunction with N. ceranae (0.71%). The active multiplication of both microsporidians was confirmed by the expression (real-time-PCR) of the N. ceranae polar tube protein 3 gene and/or the N. apis RNA polymerase II gene in 24% of the brood samples positive for Nosema spp. Both genes are related to microsporidian multiplication. As such, N. ceranae multiplication was confirmed in 1.06% of the samples, while N. apis multiplication was only observed in co-infections with N. ceranae (0.07%). Brood cells were analyzed for the presence of Nosema spp., as those are the immediate environment where the brood stages develop. The brood samples infected by Nosema spp. were in brood cells in which that microsporidians were not detected, while brood cells positive for N. ceranae hosted brood stages that were not apparently infected, indicating that this is unlikely to be the main pathway of infection. Finally, the colonies with brood infected by N. ceranae showed higher levels (numbers) of infected adult bees, although the differences were not significant before (P = 0.260), during (P = 0.055) or after (P = 0.056) brood sampling. These results show that N. ceranae is a bee parasite ubiquitous to all members of the colony, irrespective of the age of the bee. It is also of veterinary interest and should be considered when studying the epidemiology of the disease.