Results
We assayed gene expression using an array of gene fragments derived from reciprocal queen and worker larval sub-tractive cDNA libraries. Of 158 unique, consistently amplified clones isolated from these libraries, 63 showed a significant match to genes with known function. The functions assigned to these genes span approximately half of the major functional groups described by Adams et al. [10] for Drosophila melanogaster, suggesting that we have captured a fairly broad cross-section of the expressed genes in larval bees. Nevertheless, the clones derived from our libraries were biased toward particular functional groups, in a fashion that suggests a relationship between gene function and caste (Table 1). For example, we found an apparent over-representation of both ribosomal proteins and hexameric storage proteins in our libraries, relative to the expected frequencies of these gene families based on the Drosophila sequence. Of the 63 characterized clones, 15 (24%) showed significant similarity to known ribosomal protein genes. In contrast, 128 of the 6,537 annotated genes (2%) in the Drosophila melanogaster genome encode ribosomal proteins [10]. Gene knock-out experiments have implicated several of these as regulators of development rate and body size in Drosophila [11]. Assuming that ribosomal proteins help modulate ribosome activity, they may be indicators of the developmental tempo of larvae, and therefore could help pinpoint the initiation of caste determination or other major developmental events. We also cloned six apparently distinct hexameric storage proteins from the libraries, indicating a strong bias toward this family given the frequencies of hexamerins in the Drosophila genome of <<1% [10].
We raised female honeybee larvae using standard methods, then measured gene-expression patterns for larvae collected at six 24-hour intervals from the embryo stage to the middle of the fifth and final instar. A subset (n = 13) of these larvae was raised as queens, while the others (n = 14) were raised workers. Analyses of covariance in gene expression indicate three primary clusters of genes with shared expression patterns across larvae (Figure 1). These clusters define genes upregulated primarily in queen-destined larvae relative to the other larvae screened; genes downregulated in queens; and genes upregulated in young, bipotential larvae. Genes within these clusters are more similar to each other in function than expected by chance (likelihood-ratio analysis based on functional group, df = 14, X = 38.2, p < 0.001).
Genes related to RNA processing and translation showed higher rates of expression in young larvae (first and second instars) than in older larvae (Table 1). RNA helicase was strongly expressed by young larvae, as was an RNA-binding protein and translation elongation factor 2. Interestingly, two heat-shock proteins also were overexpressed in the youngest larvae. One shows a significant match to the Hsp70 family, whereas the second matches Hsp90. Heat-shock proteins are named for their ubiquitous presence following hyperthermy and other stresses [12], but members of this group have a more general role as molecular chaperones, and are involved in the assembly of newly translated proteins [13]. Hsp90 appears to be involved in the assembly of steroid hormone receptors [13]. Expression analyses on a more precise time scale, and at the level of specific tissues, should help determine whether the covariance between the expression of Hsps and agents involved in RNA processing reflects a joint role in producing essential proteins during early larval development. Queen-destined and worker-destined larvae showed broadly similar expression profiles late in the second instar, hours after queens and workers receive differential treatment in colonies. This suggests that most gene-expression changes associated with the caste programs occur after this point. Nevertheless, several genes were expressed differently at this early stage, including dihydrodiol dehydrogenase, which was expressed in lower levels by second-instar queens (normalized x = -0.067, SE = 0.025) than by either bipotential larvae (x = -0.026, SE = 0.009) or second-instar workers (x = 0.001, SE = 0.007). Dihydrodiol dehydrogenase has a general role in deactivating steroid hormones in mammals [14]. Consequently, it is intriguing that higher levels of expression of this enzyme by workers coincide with a period of decreased steroid titers in workers relative to queens. A putative lipid-binding protein also showed higher expression in workers than in queens from the second instar onward.
By the third instar, queen and worker larvae showed many differentially expressed genes (Figures 1,2) and, in each case, retained these different levels of expression through the remaining two instars. A cytochrome P450 (from the CYP4 subfamily) was more strongly expressed by workers than by queens. Members of the CYP4 subfamily have been implicated in the downregulation of steroid levels [15,16] although this subfamily is involved in a diversity of additional functions related to the metabolism of endogenous and exogenous substrates [17]. As a group, hexameric storage proteins were expressed at significantly higher levels in workers than in queens (Table 1), although a single hexamerin, from clone 1CB6 [3], showed nearly equal levels of expression in queens and workers. Hexamerins have a nutrient storage role during insect development, by bundling amino acids that accumulate during larval development for use during metamorphosis or by the adult insects. The accelerated growth rates of queens may limit their ability to allocate resources to hexamerin storage during the larval stages we assayed, as shown for Manduca sexta larvae [18]. Other genes consistently overexpressed by workers include an oxidoreductase, a fatty-acid-binding protein, aldehyde dehydrogenase and a second lipid-binding protein.
Queens overexpressed two genes directly linked to increased metabolic rates - the nuclear-encoded mitochondrial protein ATP synthase and the mitochondrial gene cytochrome oxidase I. Higher expression of two mitochondrial genes was reported previously [6], and was suggested to be an indicator of higher metabolic respiration in queen versus worker larvae. Interestingly, increased expression of these genes by queen larvae in our study was not apparent until the fifth instar in the bees we followed, a fact consistent with a role in caste-biased respiration. During the final instar, queens show the most extreme differences in growth rates, relative to workers. Queens also showed relatively high expression of a set of structural (cuticular) proteins, a histone acetyltransferase bromodomain, an ortholog to the Smcx/y mammalian sex-differentiation gene, and pyruvate dehydrogenase (Figures 1,2).