Discussion

No simple explanations for the cause of CCD emerge from the microarray analysis. Expression of detoxification and immune gene transcripts, which would indicate toxins or disease as the cause of CCD, was largely unchanged. However, considerable geographic variation existed among CCD bees, with west coast bees seemingly more severely affected. Transcripts of genes related to basic cellular processes involving ribosomal and mitochondrial function were generally less abundant in west coast bees.

P450s and COEs play multiple metabolic roles and are particularly important in the detoxification of natural and synthetic toxins in bees and other insects (20). However, the few genes in these superfamilies differentially transcribed with CCD likely serve functions other than detoxification. Expression of CYP4G11 was elevated in CCD bees, but the function of this P450 remains uncharacterized. Its ortholog in D. melanogaster, CYP4G15, is expressed only in the central nervous system, where it may be associated with ecdysteroid metabolism (21). The single COE overexpressed in CCD bees, GB10854, differs from other insect COEs in terms of its catalytic site, such that it may be incapable of detoxificative carboxylester hydrolysis (22).

Although 88 known immune-related genes were included on the microarray, these also were mostly unchanged in expression in CCD bees. Given the association between pathogens and CCD (3), the finding that so few immune genes were differentially expressed in CCD bees was unexpected. Insect guts can produce antimicrobial peptides, including the apidaecins in honey bees (23), yet CCD-related changes in transcription of these genes were not detected. This lack of response does not rule out viral infection, however, because antimicrobial proteins are ineffective against viruses. In D. melanogaster, fungal or bacterial infection induces antimicrobial peptides, but flies infected with Drosophila C virus (DCV), another picorna-like virus, did not produce more antimicrobial peptides (24) and demonstrated elevated transcription of only a small number of immune genes relative to fungal and bacterial infection (25).

Many of the transcripts differentially expressed in CCD bees were detected by probes based on ESTs matching no existing annotation (10, 11). Of the 2,729 geneless ESTs on the array, 28 were differentially expressed in CCD bees. Of all of the ESTs on which probes were based, 13 were discovered to contain rRNA sequence, and 5 of these were more highly expressed in CCD bee guts. These rRNA-coding ESTs were not identified as rRNA previously, because the A. mellifera rRNA sequence is not available in online repositories, and identification of these probes was accomplished only by manually transcribing the published rRNA sequence from printed text (26). Had these ESTs been previously identified as rRNA, the associated probes likely would have been excluded from the microarray.

The presence of rRNA on these microarrays is surprising, because both the EST projects on which the probes were based and the microarray hybridization rely on a polyadenylated 3′ tail for the initial priming step in the reverse-transcription reaction. Ribosomal RNA is transcribed by RNA polymerase I, and the resulting transcript is not polyadenylated; as such, oligo(dT)-based reverse-transcription reactions should not reverse-transcribe rRNA. Yet transcripts of rRNA have long appeared in EST projects, and their reverse transcription has been attributed to poly(A)-rich internal sequences hybridizing to the oligo(dT) or to genomic DNA contamination (27). This differential expression of polyadenylated rRNA transcripts in insects currently defies technical explanation; differential expression of these transcripts associated with age and caste in a termite (Reticulotermes flavipes) EST project (28) suggests an underlying biological explanation.

One possible explanation for the presence of poly(A) rRNA sequences in bees is that they are degradation intermediates. Polyadenylation is a well-known marker of RNA degradation in bacteria and organelles (29), and poly(A) addition is known to contribute to eukaryotic rRNA degradation (30). Polyadenylated 25S rRNA comprises ≈0.02% of all of the 25S rRNA transcripts in fission yeast Schizosaccharomyces pombe (31). Another explanation for these sequences is the “ribosomal filter hypothesis,” in which conventional mRNA transcripts include tens to hundreds of nucleotides similar to rRNA sequence in either the sense or antisense direction (32). Transcripts of genes containing rRNA sequence can bind to ribosomal subunits and alter translation efficiency (33). Short sections of rRNA function as an internal ribosome entry site (IRES) to interact with the ribosome and alter translation. Indeed, functions have been assigned to some mRNAs containing large sections of rRNA sequence (34), although none of the predicted genes annotated from the honey bee genome contains sufficient rRNA sequence to hybridize to any of the rRNA probes on the array (Table S1). A related hypothesis posits that long sequences of rRNA-like transcript may aid in protein folding, taking on part of the enzymatic activity of rRNA (35).

The picorna-like virus DWV was more abundant in CCD bees analyzed in the microarray experiment and a broader sampling of bee colonies revealed that CCD bees carry a larger number of different picorna-like viruses (Table 3). Picorna-like viruses, including IAPV and KBV, that have been associated with CCD (3) initiate transcription of viral proteins by the ribosome through an IRES rather than through the 5′-methylation cap that initiates translation of most mRNA (36). The IRES sequences from both DWV and Varroa destructor virus 1 effectively enhance translation of a reporter gene (36). Picorna-like viruses in mammals also halt translation of host mRNA through cleavage of the translation initiation factor eIF4G and the poly(A)-binding protein (37), leaving the ribosomes incapable of binding host mRNA and giving the viral RNA little competition for its IRES-mediated binding to host ribosomes (38).

While there is no direct evidence that elevated poly(A) rRNA is a result of picorna-like viral infection, one consequence of the viral disabling of ribosomal function may well be increased ribosomal degradation. Picornavirus infection in mammals both reduces protein production and causes strings of translating polyribosomes to break down, and these idle ribosomal subunits may be more susceptible to degradation (39). Ribosomal degradation also could occur in CCD bees independent of viral infection; for example, starving bacteria demonstrate elevated ribosomal degradation (39). Regardless of the cause, the abundance of poly(A) rRNA may be a useful diagnostic marker for determining CCD status, although the presence of poly(A) rRNA does not in itself indicate CCD, because healthy bees, including those used to generate EST libraries, contain these fragments.

Although gene transcript analysis did not clearly identify a specific cause for CCD, our study documents several patterns suggestive of a causal mechanism. The reduced protein synthetic capabilities that would accompany ribosomal hijacking by multiple picorna-like viruses would leave bees unable to respond to additional stresses from pesticides, nutrition, or pathogens. Although any interpretation of the presence of these rRNA fragments is speculative, the reported interaction between bee picorna-like viruses and rRNA is suggestive of a possible root cause of CCD.

To establish a causal relationship, the quantitative association between multiple picorna-like virus infections and polyadenylated rRNA fragment abundance merits further exploration. In addition, the consequences of viral infection and CCD on the function of ribosomes should be explored through assays of translational efficiency. Because of the potential for translational interference, studies on immune suppression should focus on bioassays or protein abundance rather than on immune gene transcripts.

Our results also indicate an unappreciated variation in gene expression patterns and pathogen loads with geography. This variation provides insight into the different stresses facing bees and clearly demonstrates that diagnostic surveys must sample extensively across numerous bee populations. Colony surveillance via assay of rRNA-like transcript abundance may provide an earlier indication of CCD status than has hitherto been available and allow beekeepers to take actions to reduce losses.