The nutrient amino acid transporter slimfast plays an important role in nutritionally dependent processes, such as coordinated growth in both males and females and oogenesis in females. By acting as a nutrient sensor, slimfast stimulates processes like nutrient import, metabolism, and translation in response to nutrient availability via the Target of Rapamycin (TOR) signaling pathway [21, 22]. TOR signaling activation relies especially on slimfast expression in the insect fat body, evidenced by the fact that in Drosophila melanogaster, downregulating slimfast in fat body cells disrupts overall growth . In mosquitoes, slimfast expression in the fat body also activates vitellogenesis , a key process in oogenesis. While oogenesis is reproductive in nature, it is fundamentally nutrient-driven because female reproductive processes, unlike male reproductive processes, are energetically costly and depend directly on nutrient availability [24, 25]. Although a male-specific role for slimfast has never been documented in any insect, we present evidence that some paralogs in the aphid-specific slimfast expansion have evolved a male functional role.
Aphids possess male-enriched nutrient amino acid transporters, including conserved male-biased paralogs in the aphid-specific slimfast expansion
We have identified five nutrient amino acid transporters with at least 4-fold enriched expression in males relative to at least one female aphid morph. Although the software Blast2GO failed to annotate all five of the contigs we identified from the microarray (Table 1 and Additional file 3), InterProScan IDs and Pfam searches provided strong support that all five contigs are amino acid transporters and that four of the five contain either the Aa_trans or AA_permease transmembrane domains (Table 1). These domains characterize two amino acid transporter families, the amino acid/polyamine/organocation (APC) family (TC #2.A.3), and the amino acid/auxin permease (AAAP) family (TC #2.A.18) [26–29]. These amino acid transporter families contain all but a handful  of the known nutrient amino acid transporters. Though one contig (8321) did not significantly match any Pfam domain (Table 1), its strongly supported phylogenetic position within the A. pisum expansion of slimfast (Figure 1), a member of the APC family, indicates that contig 8321 is also a member of the APC family. Of the five male-enriched nutrient amino acid transporters (Table 1 and Additional file 3), contigs 1492 and 8321 (see Additional file 4 for GenBank accessions) are male-biased, being overexpressed in males relative to all three female reproductive morphs.
Shifting our study focus to A. pisum supported a conserved male role for slimfast. The phylogenetic placement of contigs 1492 and 8321 indicates that they are orthologous to A. pisum slimfast paralogs ACYPIslif6 and 5 respectively (Figure 1). Male-biased expression in these paralogs (Figure 2) suggests that their male-biased expression is conserved minimally in the Macrosiphini tribe, of which A. pisum and M. persicae are members. Further, QPCR expression analysis revealed one more male-biased paralog, ACYPIslif10 (Figure 2). Male expression in ACYPIslif10 was low compared to the expression observed for ACYPIslif5 and 6 (Figure 2), which may indicate that it is expressed only in a particular tissue and its true expression level is confounded by quantifying expression in whole insects. Because slimfast is not known to have a male-specific role, the presence of male-biased slimfast paralogs in aphids is puzzling. If aphid slimfast paralogs retain a function in activating TOR signaling and nutrient-dependent processes, then the presence of male-biased paralogs could indicate that male and female aphids must overcome different nutritional challenges.
The evolutionary origin of male-biased slimfast expression in aphids
Given the phylogenetic distribution of male-biased expression (Figure 2), we are unable to conclusively reconstruct when male-biased expression evolved within the slimfast expansion. The current data set can explain the distribution of male-biased expression by two alternatives. First, male-biased expression could be derived, which could have happened in one of two ways. Either male-biased expression evolved independently for each paralog, or it evolved twice independently and was lost in the lineage leading to ACYPIslif4. A second, equally parsimonious, explanation is that male-biased expression is the ancestral state and was lost three times. Derived and ancestral male-biased expression in the expansion are thus equally plausible given our data. Distinguishing between these two explanations would be facilitated by data for the three paralogs lacking expression information (Figure 2).
Despite the lack of resolution in our results for when male-biased expression evolved in the slimfast expansion, support for derived male-biased expression in aphids can be gleaned from the literature. In a study examining sex-biased genes in seven Drosophila species, slimfast was significantly female-biased in five species and not significantly enriched in either sex for the other two species (see Supplementary Tables from ). This expression may reflect the ancestral state of the aphid slimfast expansion since Drosophila genomes have only one slimfast copy (http://phylomedb.org), and the essential role of slimfast in growth  strongly suggests that Drosophila slimfast is under strong purifying selection. Our molecular evolution analyses support strong purifying selection for Drosophila and other insect slimfast, evidenced by the significantly lower ω found for branches outside the aphid slimfast expansion (Table 3). Further, the known female and sex-neutral roles [18, 23] but lack of documented male role suggests that male-biased expression (and corresponding male function) is derived among aphids.
Evolution of a male-biased functional role in aphid slimfast
Male-biased genes reflect traits that increase male fitness, such as male-male competition, sexually selected characters and secondary sexual characters . The male roles of three aphid slimfast paralogs can be hypothesized from our current and previous results. Although we did not examine tissue-level expression in this study, our previous work  quantified relative expression levels of slimfast paralogs in asexual female head, gut and bacteriocytes. While tissue-level expression patterns may not be identical in both sexes, these data provide a framework within which to formulate some of the possible testable hypotheses about the function of male-biased paralogs. In this context, all three male-biased paralogs show highly enriched expression in asexual female gut , consistent with slimfast expression in Drosophila  and Tribolium [Supporting Table 3 from ].
The digestive tract interfaces between an animal and its diet, playing a critical role in uptaking dietary nutrients. Dietary nutrient availability is central to the aphid/Buchnera nutritional symbiosis because deficient dietary amino acids must be synthesized by Buchnera. While Buchnera-containing bacteriocytes are abundant in females, males contain relatively few  and in extreme cases, males completely lack bacteriocytes [35, 36]. This sex-based difference in a major nutrient provisioning cell type suggests that males and females differ in their ability to synthesize amino acids deficient in their diet. Differential amino acid biosynthesis could be compensated for by upregulating amino acid transporters in the male gut, enabling greater uptake of certain amino acids from phloem sap.
In contrast, other expression patterns we observed previously suggest an alternative possibility for male function. In addition to being enriched in gut, two of the male-biased paralogs (ACYPIslif6 and ACYPIslif10; Figure 2) were also enriched in the asexual female head . These paralogs could thus function in any of the head tissues included in the analysis, such as brain, eyes, mouthparts, antennae, or salivary glands. These tissues suggest that the male-biased paralogs could be implicated in sensory functions, such as locating a mate.
The possibility remains that the male-biased paralogs have evolved a different expression pattern (and function) from asexual females. One possible role for these male-biased paralogs that we cannot predict from female expression profiles is a role in male reproduction, such as spermatogenesis. Although slimfast has never been implicated in spermatogenesis, related mammalian transporters (SLC7A1 and SLC7A2) deliver L-Arginine to rat seminiferous tubule cells , where spermatogeneis begins. Thus, divergence of some slimfast paralogs to fill a male reproductive role is plausible. In light of the molecular evolution results, paralog ACYPIslif10 is the best candidate for having a role in spermatogenesis since its terminal branch has experienced an extremely accelerated rate of non-synonymous substitutions (Table 3). Accelerated evolutionary rates are commonly associated with male-biased genes, especially genes involved in sperm competition . While the accelerated ω we observed is consistent with both positive and neutral/relaxed selection, both types of selection can lead to functional divergence [38, 39]. Additional studies can tease apart the various hypotheses we have presented for the role played by male-biased slimfast paralogs in aphid biology.
Insights on the maintenance of duplicate amino acid transporters in aphids
By pointing towards a derived evolutionary origin of male-biased function in slimfast, our results provide insights into a fundamental question of how the aphid genome retains the slimfast expansion and other nutrient amino acid transporter duplications [see also 9]. Given that most gene duplications fail to reach fixation , it is intriguing that the aphid genome possesses more nutrient amino acid transporters than other sequenced insects . The presence of these duplicate amino acid transporters indicates that there must be a selective advantage to their maintenance. As mentioned above, we previously discovered that some duplicate nutrient amino acid transporters (including some slimfast paralogs) are highly enriched in bacteriocytes, leading us to hypothesize that these transporters mediate nutrient exchange across the A. pisum/Buchnera symbiotic interface . A role in mediating endosymbiotic interactions strongly suggests that duplicate nutrient amino acid transporters and the slimfast expansion are maintained in the genome because they diverged to fill a novel role in symbiosis. The current study supports a different, but not mutually exclusive, hypothesis that the slimfast expansion (and possibly other nutrient amino acid transporter duplicates) is maintained because some paralogs diverged to fill novel, sex-specific roles. Future studies will be able to test the relative significance of symbiosis and sex in maintaining amino acid transporter gene duplications by examining the genetic architecture and expression of nutrient amino acid transporters in other phloem-feeding insects with different, less complex life cycles.