Iridovirus and microsporidian linked to honey bee colony decline

Abstract

Background: In 2010 Colony Collapse Disorder (CCD), again devastated honey bee colonies in the USA, indicating that the problem is neither diminishing nor has it been resolved. Many CCD investigations, using sensitive genome-based methods, have found small RNA bee viruses and the microsporidia, Nosema apis and N. ceranae in healthy and collapsing colonies alike with no single pathogen firmly linked to honey bee losses.

Methodology/principal findings: We used Mass spectrometry-based proteomics (MSP) to identify and quantify thousands of proteins from healthy and collapsing bee colonies. MSP revealed two unreported RNA viruses in North American honey bees, Varroa destructor-1 virus and Kakugo virus, and identified an invertebrate iridescent virus (IIV) (Iridoviridae) associated with CCD colonies. Prevalence of IIV significantly discriminated among strong, failing, and collapsed colonies. In addition, bees in failing colonies contained not only IIV, but also Nosema. Co-occurrence of these microbes consistently marked CCD in (1) bees from commercial apiaries sampled across the U.S. in 2006-2007, (2) bees sequentially sampled as the disorder progressed in an observation hive colony in 2008, and (3) bees from a recurrence of CCD in Florida in 2009. The pathogen pairing was not observed in samples from colonies with no history of CCD, namely bees from Australia and a large, non-migratory beekeeping business in Montana. Laboratory cage trials with a strain of IIV type 6 and Nosema ceranae confirmed that co-infection with these two pathogens was more lethal to bees than either pathogen alone.

Conclusions/significance: These findings implicate co-infection by IIV and Nosema with honey bee colony decline, giving credence to older research pointing to IIV, interacting with Nosema and mites, as probable cause of bee losses in the USA, Europe, and Asia. We next need to characterize the IIV and Nosema that we detected and develop management practices to reduce honey bee losses.

Figure 1. Discriminant Function Analysis for differences in pathogen peptide counts among strong, failing, and collapsed honey bee colonies.

Function 1 explains 81 percent of discriminating variance and contrasts higher incidence of iridovirus (IIV), Nosema, and to a lesser extent BQCV in failing colonies with higher incidence of DWV and some IAPV in the remaining groups. Vertical and horizontal lines mark the non-CCD out-group as a reference set.

Figure 2. Decline in forager flights in conjunction with increasing counts of iridovirus (IIV) and Nosema peptides.

The peptides were detected in dead worker honey bee samples collected from a single collapsing observation hive at the University of Montana, Missoula. Forager flights are absolute counts per day, tabulated by an automated honey bee counter. Peptide counts are the summed counts by day of collection for all unique IIV Nosema peptides in each sample. We suspended sampling in mid-August, as the bee population was too weak to sample without affecting the colony. We collected the queen and last few bees for a final sample in early September. Sample size varied from about ten bees to more than 100 per sample interval, depending on how many dead bees were obtained from the entrance tube.

Figure 3. Survival over a fourteen day post-infection period observed in cage-trials of Nosema ceranae and IIV infected honey bees.

Newly emerged, 1–3 day old, bees were used in all experiments. Figure represents the combined survival results for 4 biological replications (N = 30 bees in each group for each biological replicate). Bees that perished within 24 hours of inoculation were not included in the survival curve analyses. Deaths in control group were confirmed not to be pathogen related via mass spectroscopy analyses. Inoculum sizes and doses described in materials and methods section. Log Rank Tests Kaplan-Meier Curve Analyses: 1) Control VS. N. ceranae - P = 0.01, 2) Control VS. IIV alone – P<0.01 (0.008), 3) Control VS. Nosema + IIV – P<0.01 (0.0001), 4) Nosema alone vs. IIV alone – P = 0.90, 5) Nosema alone VS. Nosema + IIV – P = 0.04, 6) Virus alone VS. Nosema + IIV – P = 0.04. These results strongly suggest that the combination of N. ceranae and IIV is associated with increased bee mortality.