MS-based proteomics provided an unrestricted and unbiased approach for surveying pathogens in honey bee colonies. Our results detected a DNA virus and two RNA viruses that had not been previously reported in honey bees from the USA. The potential correlation of IIV with CCD may previously have gone unnoticed because these are large DNA viruses, not the small RNA viruses commonly considered to be the cause of most bee diseases. The correlation between IIV and N. ceranae for bee colonies exhibiting CCD implies that they track each other.
Interestingly, the presence or absence of IIV in a given honey bee colony may explain why in the USA N. ceranae
sometimes seems to contribute to severe colony losses (IIV present), and sometimes not (IIV absent), as reported both by researchers and beekeepers 
. The mechanism by which these two pathogens interact to potentially increase bee mortality is unknown. It may be that damage to gut epithelial and other host cells by the N. ceranae
polar tube allows more robust entry of the virus. Alternatively, replication of N. ceranae
in honey bee cells may cause a decrease in the bees' ability to ward off viral infections that normally could be controlled. These types of studies await isolation of the definitive virus.
Other than the presence of the IIV, only the concurrent absence of Deformed wing virus, another RNA virus, was significant with respect to CCD. We recognize that the iflaviruses VDV-1 and Kakugo virus appear to be variants of Deformed wing virus, whereas the dicistroviruses Kashmir bee virus, Acute bee paralysis virus and IAPV are also closely related to one another 
. Even closely related viruses may express different etiologies, and as such, we treated them as separate variables in our statistical analyses.
Virtually all of the bees from CCD colonies contained Nosema
species and IIV, whereas IIV was not found in bees from packages imported from Australia nor in bees from colonies of the non-migratory, commercial bee operation in Montana. CCD has not been reported by the Australians 
or the Montana beekeeper.
Since inapparent non-lethal infections by IIVs are common 
, detection of IIV in some strong colonies and in the remnant young bee populations of collapsed colonies is to be expected. Presence of IIV in bees located in strong colonies may indicate a non-lethal infection, an early stage of the disease, or possible resistance to the virus in these colonies. In collapsed colonies, the presence or absence of IIV in remnant young bee populations is likely dependent upon the extent of infection in the colony and unknown pathophysiological factors that may affect persistence, colonization, and replication of the virus. Development of a PCR- or antibody- based assay to detect IIV in honey bees will allow us to rapidly detect and track the presence of IIV throughout the honey bee life cycle in a given colony.
Large amounts of IIV in failing colonies is consistent with an infection that proliferates in bees but not necessarily to a degree that results in the iridescent coloration of infected bee tissues that is characteristic of IIV disease. The sustained high levels of IIV and Nosema
peptides that occurred in the observation colony as the frequency of forager flights declined strongly implicates an IIV-6-like virus and Nosema
as co-factors in CCD, since forager bee activity declined as pathogen loads peaked. In Spain, researchers have published studies linking N. ceranae
to colony collapses in that country, yet we noticed that pest and disease surveys have reported the presence of an iridescent virus in colonies surveyed for mites and diseases 
Because of their virulence, IIVs have been investigated as candidates for use as biopesticides 
. Invertebrate iridescent viruses (IIVs) are large, icosahedra, double-stranded DNA viruses 
. Of the many isolates reported from insects, only two, IIV-3 and IIV-6 
have been subjected to complete genome sequencing and an additional 24 have been partially characterized 
. Historically, IIVs were numbered according to date of isolation 
. Uniformly packed particle arrays 
of these viruses produce opalescent colors in the tissues of heavily infected hosts, particularly in insects in damp or aquatic habitats. These viruses have been shown to alter insect growth, longevity, and reproduction, and induce cell apoptosis 
. In silkworms, IIV-1 can induce epidermal tumors 
Patent IIV infections are almost invariably lethal but inapparent or covert infections may be common 
. Inapparent infections may not be lethal, but may affect the reproduction and longevity of covertly infected hosts 
. IIV-3 is thought to be restricted to a single species of mosquito 
, although we found peptides close to those of IIV-3 in bees from an observation hive. These bee samples were hand-picked with forceps, so we are confident that our observation hive samples did not include mosquitoes or other insects.
Other IIVs, such as IIV-6, naturally infect various species of Lepidoptera and commercial colonies of Orthoptera. There is evidence that hymenopteran endoparasitoids can become infected if they develop in an infected caterpillar 
. IIVs have also been studied for control of mosquitoes 
and boll weevil 
, the latter work examined the virus itself, with an emphasis on the proteins that it produces as the basis for a possible biopesticide. A U.S. Patent has already been awarded 
There is one known iridescent virus in bees. IIV-24, originally isolated from the Asiatic honey bee Apis cerana
, severely affects bee colonies, causing inactivity, crawling, and clustering disease 
. Proteomics could not identify IIV-24 in any of our samples because there are no IIV-24 sequences in the current databases. Thus, thus the identity of the IIV in our samples remains undetermined.
Based on the sequence data generated from MSP, the IIV identified appears to be closely related to IIV-6, possibly because this is the only IIV in the Iridovirus
genus that has been completely sequenced. The major capsid protein represents approximately 40 percent of the total particle polypeptide and is highly conserved, so sequencing peptide fragments may frequently identify IIV-6 as being the most likely candidate 
. This argument is reinforced by some results coming back as IIV-3, which is presently assigned to a different genus in the family (Chloriridovirus
) and only reported to occur in a mosquito 
There is little information about IIVs in bees, although there are historical reports associating IIVs with severe bee losses in India 
, the U.S. 
, and possibly Spain 
. In the 1970s, in northern India, almost every bee was infected with IIV-24, with 25–40 percent annual colony loss 
. The disease was manifested by inactivity, clustering, and crawling sickness.
Transmission of IIV-24 is suspected to occur via eggs, feces, or glandular secretions in food 
. Evidence that IIV-24 was the cause of Indian bee losses was based on turquoise and blue iridescence seen in affected bees and tissues, serological tests, and microscopic examination of sick bees. IIV was the only recognizable parasite in all samples. IIV-24 was strongly correlated with co-infective Nosema
species and tracheal mites in diseased colonies of Apis cerana 
. Tracheal mites were found in some, but not all of the sick colonies 
. The fat body was always attacked by the virus, and other tissues and organs, including the ovaries were frequently infected 
In addition, an iridescent virus has also been associated with mites, which may act as vectors, and has been implicated in bee losses in the United States. While investigating unusually high losses of bees in the northeastern United States, Camazine and Liu 
extracted a putative iridovirus from Varroa
mites collected from a colony that perished four weeks later. They concluded that viral transmission within the colony might kill both mites and bees, but they were not able to discover the virus in time to determine whether bees in the colony were infected, and they were unable to purify the virus or determine whether the virus could be transmitted to bees by inoculation.
One or more species of external mites were suspected of being carriers of the virus in Indian bees 
, as was also the case in the U.S., with Varroa
acting as the vector 
. The need for a better knowledge of the ecology of IIVs has been emphasized in order that preventive measures could be taken to not only offset damage to Apis cerana
but also to reduce the chance that Apis mellifera
could become infected by this pathogen 
. Indeed, IIV-24 was experimentally inoculated and found to lethally infect A. mellifera
, forming cytoplasmic quasi-crystalline aggregates of virus particles in cells of the fat body, hypopharyngeal glands, the gut wall, and proximal ends of the Malpighian tubules 
These historical findings of IIV, mites, and Nosema
species are intriguing since researchers studying both Nosema ceranae
and CCD in Spain observed IIV-like particles in bee samples by electron microscopy 
. U.S. investigators studying CCD observed structures in thoraxes of bees described as ‘peculiar white nodules’, resembling tumors, that contained crystalline arrays 
, similar to those described for IIV infections. Also, it appears that the IIV-6 genome encodes for one or more polypeptides that can produce insect mortality by inducing apoptosis without the need for viral replication 
The suspected source of Nosema ceranae
in Apis mellifera
is the Asian bee Apis cerana 
. This bee species is also known to be infected by Thai sacbrood virus and by Kashmir bee virus. Kashmir virus was first detected as a contaminant in a sample of iridescent virus from India, as well as IIV-24 
. The same virus was linked to bee losses in Canada in the early 1990s 
. This suggests that perhaps not only the microsporidium N. ceranae
, but other pathogens as well may have jumped from Apis cerana
to Apis mellifera
, as predicted by Bailey and Ball in 1978 
It also implies that if Kashmir bee virus has been in North America for more than twenty years, so might IIV and Nosema ceranae
. That would fit the time line of the first observations of this complex of pathogens, and of severe bee losses in India in the 1970s. It also leads us to ask whether the first widespread losses of bees in the USA, described as Disappearing Disease in the 1970s 
, may have been early outbreaks of CCD.
Our own work, described here, provides multiple lines of correlative evidence from MSP analysis that associate IIVs and Nosema with CCD in the USA. We conclude with results of laboratory inoculations of caged bees with IIV and Nosema that demonstrate the potential for increased lethality of mixed infections of these two pathogens. Our study strongly suggests a correlation between an iridescent virus, Nosema, and CCD. Our inoculation experiments confirmed greater lethality of an IIV/Nosema co-infection compared to infections involving each pathogen alone. Future research using the specific strains of IIV isolated from infected bees will surely confirm whether a synergistic or additive interaction between these two pathogens results in the signs and symptoms of CCD.
The fact that IIV-6 inoculated bees experienced increased mortality in the presence of Nosema clearly strengthens the significance of all lines of evidence pointing to an interaction between an IIV and Nosema ceranae. Lack of a stronger effect by preparations containing IIV-6 may be due to the possibility that the IIV detected by proteomics is either a strain of IIV-24 or a strain of IIV-6 that is more specifically adapted to honey bees, and consequently more virulent than the strain of lepidopteran origin used in our inoculation experiments. It is, of course, critical to isolate the IIV from CCD populations, compare it to known IIVs and particularly IIV-24, and then challenge CCD populations with this strain. This work is in progress.
Moreover, we used a fairly low dose of IIV-6 and Nosema ceranae spores. For example, IIVs are generally not highly infectious by ingestion. Similarly, virulence studies on N. ceranae have reported using over 200,000 spores per bee in cage trials whereas we used a four-fold lower dose. It will also be interesting to test whether the interaction between IIV and N. ceranae is specific, or a general “stress” phenomenon that could also be reproduced by addition of N. ceranae and any additional bee virus.
In our studies, we applied six independent scenarios to the assessment of potential causes or markers of CCD and got the same answer, giving us confidence in the results, since this inference approach is approximately analogous to applying the same technique to six different assessments 
. Our results also provide credibility to older, often overlooked work by others that associated IIV with bees, tracheal and Varroa
species and severe bee losses. In our samples, Varroa
mites were seen in many CCD colonies, but not in all. Importantly, our limited results do not completely fulfill the requirements of risk characterization, nor do they clearly define whether the occurrence of IIV and N. ceranae
in CCD colonies is a marker, a cause, or a consequence of CCD. Our findings do make a strong case for a link between an Iridescent virus and Nosema
with CCD and provide a clear direction for additional research to answer these questions.
We anticipate that there also may be questions as to why IIV was detected in our study, but has not been found in any current published research on CCD. And, if these viruses were present, why weren't they seen in infected tissues of the European honey bee, Apis mellifera?
First, iridescent viruses have been seen before in Apis mellifera
, both in Europe and in the USA. Researchers in Spain reported seeing iridescent virus in honey bees 
, and Camazine 
saw a putative iridescent virus in Varroa
mites following a collapse of colonies in the northeastern part of the U.S. in the 1990s. Also, inapparent infections by iridescent viruses may involve a low density of IIV particles in infected host cells 
, so without sensitive techniques such as MSP, it is not surprising that infections in CCD bee colonies were previously missed.
The large number of IIV proteins that we identified, 139 in all, represent a significant fraction of the total IIV proteome. The recently published genome for IIV-6 
suggests a total proteome of 137 unique proteins. The 139 polypeptides identified for the IIV strain in our study must therefore represent a near complete sample of the total viral proteome belying any criticism that our identification of IIV may be a spurious consequence of accidental matching of a few peptide fragments.
We conclude that the IIV/Nosema association may be critical in honey bee mortality linked to CCD. Although viral diseases are currently manageable only by culling, Nosema infections are treatable with several current management techniques. We suggest that for beekeepers suffering from colony losses, disruption of the potential IIV/Nosema relationship using treatments that are available to control Nosema species may be one option to help reduce honey bee mortality. Again, whether this identified bee IIV and its potential interaction with Nosema species is the cause or marker of CCD, is unknown, but our results clearly suggest that further research in this area is urgently required.