Nosema ceranae spillover from Apis mellifera to an Australian stingless bee; Tetragonula hockingsi, Far North Queensland, Australia, 2016

Brief description

This dataset includes data obtained from laboratory experiments and field surveys to investigate the susceptibility of Tetragonula hockingsi to Nosema ceranae, prevalence of infection, and transmissibility via flowers (Sphagneticola trilobata). All the data was collected from North Queensland, Australia, during 2016.

Full description

This data was collected to explore Nosema ceranae spillover from managed bees (Apis mellifera) to stingless bees (Tetragonula hockingsi), N. ceranae virulence within T. hockingsi and a mechanism of transmission.

1. Experimental infection of T. hockingsi with N. ceranae

Methods:

We collected T. hockingsi from six hives located at different sites (Table 1). Hives from different localities were chosen to ensure any local variation in populations was not a factor. We captured 120 individual returning forager bees from each hive directly into plastic bags (Code: 397786P, Multix Pty Ltd). We immediately randomly assigned 15 bees to one of eight cages per hive and randomly assigned each cage to either the inoculated, or non-inoculated (control group), thus ensuring similar capture and handling techniques were used for all bees. Cages were constructed from rectangular polypropylene 500ml containers (Product Code: BS500; BBC Plastics Pty Ltd). We kept cages in a cool, dark container until we returned to the laboratory and placed them into an incubator (TRH-460-GD, Thermoline Scientific Equipment Pty Ltd) at 27°C, 40% relative humidity, and constant darkness. After 1 hour in the incubator, the four cages assigned to the inoculation group each received 0.5µL of a 50% sucrose solution containing 75 000 N. ceranae spores on the cage floor hourly for five hours. We purified the spores using the centrifuge method described by Fries et al.2 and diluted the purified spores with 50% (1:1 weight: volume) sucrose solution to obtain a concentration of 75 000 spores per 0.5 μL. We previously established this dose would produce a detectable infection. We fed the four cages assigned to the non-inoculated group 50% sucrose solution without spores by the same method. We switched both groups to 25% sucrose solution ad libitum via a small dish in each cage. We inspected each cage daily to remove dead bees and ensure sucrose solution was freely available. All dead bees were checked individually for spores using standard microscopy techniques and PCR24. We rotated all the cages within the incubator on a daily basis, ensuring that any desiccation by fans and heating elements would affect all cages equally. No abnormal fluctuations in temperature or humidity were recorded by a data logger device placed centrally in the incubator (Model: DS1402D-DR8, Thermodata Pty Ltd). In total, we caught 720 T. hockingsi for our experiment, of which six escaped during feeding. All the escaped T. hockingsi were from different cages and equally distributed across treatment groups. Thus there were 357 bees in both inoculated, and non-inoculated groups.

Table 1. T. hockingsi hives used to collect bees for experimental infection of T. hockingsi with N. ceranae (2–7), and T. hockingsi hives monitored to ascertain N. ceranae infection status in wild populations (1–6), locality, latitude, longitude (accuracy ~10m), and elevation.

2. N. ceranae in T. hockingsi hives 

Methods:

We surveyed six T. hockingsi colonies that were located across a diverse range of habitats in the study area (Cairns region, Queensland, Australia) (Table 1). We collected 15 returning foragers from each colony monthly between April and August 2016 by catching them at the hive entrance directly into plastic bags (Code: 397786P, Multix Pty Ltd). The bees were euthanized in the bags by freezing at  20ºC, and then tested for N. ceranae spore presence using standard microscopy techniques and PCR²⁴. April and May samples were tested by grouping the 15 individual bees collected from a colony together, yielding a total spore count for the 15 bees for each hive. Bees collected from June-August were tested individually, thus yielding individual and group spores counts, plus the number of infected individual bees. N. ceranae spore presence and identification was completed using the methods as described in Spore counting and identification. 

3.Transmission of N. ceranae via flowers

Methods:

We placed five 14 day-old N. ceranae infected A. mellifera into each of 15 cages containing a single Sphagneticola trilobata (Singapore daisy) inflorescence. Cages were constructed from round polypropylene 840ml containers (Product Code: B30; BBC Plastics Pty Ltd). The inflorescence had been picked as a closed bud the previous day and placed with the stem in water into an insect-proof container to open, thus preventing any contamination of the inflorescence prior to opening. Each group of five bees was allowed to forage for three hours, after which they were removed, euthanized, and tested for spore presence using light microscopy and PCR analysis7. The flower was then removed and placed into a new clean cage, thus removing the cage as a possible source of transmission. Meanwhile we collected 150 T. hockingsi foragers from the colony in the survey in which we had never detected N. ceranae (colony ID: 1). We randomly assigned half of these, in groups of five, to the 15 cages containing an inflorescence previously exposed to the infected A. mellifera, and the other half, in groups of five, to 15 identical cages containing an inflorescence that had not been exposed to infected A. mellifera. All T. hockingsi were left in the cages for three hours, after which time they were transferred to an incubator and fed 25% sucrose solution ad libitum. After 8 days, we euthanized T. hockingsi by freezing at -20C and tested for N. ceranae spores. We also tested all flowers and A. mellifera for N. ceranae spore presence.