- Research article
- Open Access
Novel male-biased expression in paralogs of the aphid slimfast nutrient amino acid transporter expansion
BMC Evolutionary Biology volume 11, Article number: 253 (2011)
A major goal of molecular evolutionary biology is to understand the fate and consequences of duplicated genes. In this context, aphids are intriguing because the newly sequenced pea aphid genome harbors an extraordinary number of lineage-specific gene duplications relative to other insect genomes. Though many of their duplicated genes may be involved in their complex life cycle, duplications in nutrient amino acid transporters appear to be associated rather with their essential amino acid poor diet and the intracellular symbiosis aphids rely on to compensate for dietary deficits. Past work has shown that some duplicated amino acid transporters are highly expressed in the specialized cells housing the symbionts, including a paralog of an aphid-specific expansion homologous to the Drosophila gene slimfast. Previous data provide evidence that these bacteriocyte-expressed transporters mediate amino acid exchange between aphids and their symbionts.
We report that some nutrient amino acid transporters show male-biased expression. Male-biased expression characterizes three paralogs in the aphid-specific slimfast expansion, and the male-biased expression is conserved across two aphid species for at least two paralogs. One of the male-biased paralogs has additionally experienced an accelerated rate of non-synonymous substitutions.
This is the first study to document male-biased slimfast expression. Our data suggest that the male-biased aphid slimfast paralogs diverged from their ancestral function to fill a functional role in males. Furthermore, our results provide evidence that members of the slimfast expansion are maintained in the aphid genome not only for the previously hypothesized role in mediating amino acid exchange between the symbiotic partners, but also for sex-specific roles.
A fundamental goal of molecular evolutionary biology is to understand the evolutionary fate and biological consequences of duplicated genes. Most duplicate genes fail to reach fixation , strongly suggesting that retained gene duplicates provide a selective advantage. Recent genome sequencing projects have revealed that the water flea, Daphnia pulex, and the pea aphid, Acyrthosiphon pisum, are unique among arthropods in having exceptionally high levels of tandem, lineage-specific gene duplication and retention [2–9]. Apart from high gene duplication levels, both Daphnia and aphids, in contrast to other sequenced insects, are cyclical parthenogens with complex life cycles characterized by extensive polyphensim [2, 3]. The shared life cycle complexity and gene duplication levels of aphids and Daphnia suggest that polyphenism may be a major factor underlying the extensive gene duplications characterizing these two genomes.
Several aphid-specific gene family expansions have been suggested to be associated with the aphid life cycle and polyphenism . For example, several genes involved in early developmental signaling pathways have undergone aphid-specific duplications , which may account for the differences observed in embryonic development between live-bearing, asexual females and egg-laying, sexual females [4, 10]. One major developmental difference between asexual and sexual females is the type of cell division that initiates oogenesis. While sexual female oocytes undergo standard meiosis, asexual female oocytes undergo a modified form of meiosis before initiating development of a parthenogenetic embryo . The differences in cell division between asexual and sexual female oocytes might be accommodated by gene duplications found in cell cycle genes, some of which are differentially expressed in sexual and asexual female ovarioles . Additionally, since different morphs within the same clonal line are genetically identical, their morphological, biological and ecological differences must result from differences in gene expression [e.g. ], which may be facilitated by duplicate, divergent chromatin remodeling genes .
In contrast to an hypothesized association with polyphenism, some aphid-specific gene duplications appear to be more connected with other aspects of aphid biology, such as their diet. Aphids feed on plant phloem sap, a diet rich in sugar, which creates a high positive osmotic potential between the ingested phloem sap and the aphid hemolymph. This osmotic challenge must be overcome in order for aphids to avoid desiccation [8, 13]. Aphids cope with their osmotically challenging diet both by voiding excess sugar as liquid waste called honeydew , and by transporting sugars across the gut epithelium into their hemolymph via sugar transporter proteins . Interestingly, aphid sugar transporters have undergone aphid-specific gene duplications , which may enable aphids to utilize a sugar rich diet.
Another group of diet-associated genes that have undergone aphid-specific duplications are the nutrient amino acid transporters . Duplications in nutrient amino acid transporters may be explained by another feature of the aphid phloem sap diet. Apart from being rich in sugar, phloem sap is also deficient in essential amino acids. Essential amino acids cannot be synthesized de novo by metazoans, so their deficit in phloem is compensated for by the intracellular bacterial symbiont, Buchnera aphidicola [15, 16]. Buchnera reside within membrane-bound compartments in specialized aphid cells called bacteriocytes, where they exchange non-essential amino acids from the aphid for the essential amino acids they synthesize [reviewed in ]. Recently, we discovered that some nutrient amino acid transporters are highly expressed in bacteriocyte cells where Buchnera reside, suggesting that these amino acid transporters mediate amino acid exchange between aphids and Buchnera . Our finding that bacteriocyte-enriched amino acid transporters are often members of gene family expansions suggests that past duplication events enabled some duplicates to conserve their ancestral function while others diverged to fill a role in symbiosis .
Here we propose that in addition to a role in symbiosis, duplicate nutrient amino acid transporters have evolved sex-biased roles. In microarray data for the aphid species Myzus persicae, we unexpectedly discovered that two nutrient amino acid transporters have highly male-biased expression. These transporters, orthologous to the Drosophila gene slimfast, were nested within an aphid-specific slimfast expansion among 10 A. pisum paralogs. Male-biased expression for these two paralogs is conserved in A. pisum and detailed expression analysis in A. pisum revealed one additional male-biased slimfast paralog and one asexual female-biased slimfast paralog. Together, these data suggest that, in addition to the symbiosis-based functions proposed by Price et al., , the aphid-specific slimfast expansion is maintained for sex-biased functions.
Microarray and automated annotation results
We mined a microarray comparing expression between male, sexual female, and asexual female Myzus persicae aphids for genes with differential expression between males and at least one female morph (i.e. genes that were not sex-neutral). Comparisons were made with two types of asexual females: asexual females from perpetually parthenogenetic cultures requiring higher temperatures and long day lengths (long day asexual females) and asexual females collected from sexual cultures, induced by lower temperatures and shortened day lengths (short day asexual females). Comparing males and sexual females to both long and short day asexual females enabled us to control for day length effects in gene expression (since sexual morphs are only present under short day conditions).
Genes with differential expression between males and at least one female reproductive morph are referred to as "male-enriched" or "female-enriched". These sex-enriched genes were identified by fold change (4-fold or greater upregulation) and significance (False discovery rate (FDR) < 0.05). Of the 10,478 M. persicae unigenes represented on the microarray, 768 (~7%) were male-enriched and 725 (~7%) were female-enriched (Additional file 1; a list of all sex-enriched genes with descriptions, annotations and fold changes can be found in Additional files 2 and 3). Of the male-enriched genes, 126 (16%) were overexpressed in males relative to all three female morphs and of the female-enriched genes, 82 (11%) were enriched in all three female morphs relative to males. We refer to genes that are differentially expressed between males and all three female reproductive morphs as "male-biased" or "female-biased". One gene was enriched in both sexes depending upon the female morph: contig 2728 (see Additional file 4 for corresponding EST accession numbers) was overexpressed in males relative to sexual females and overexpressed in asexual females relative to males (Additional files 1, 2, and 3).
Mapping and annotation results obtained from Blast2GO are summarized in Additional file 1. The homology search returned significant BLAST hits for 501 (65%) male-enriched and 554 (76%) female-enriched genes. Gene Ontology (GO) terms were found for 371 (48%) male-enriched and 414 (57%) female-enriched genes. Round one annotation, which excluded electronic annotations, annotated 202 (26%) male-enriched and 291 (40%) female-enriched genes. Round two annotation, which included electronic annotations, annotated an additional 63 (8%) male-enriched and 51 (7%) female-enriched genes. InterProScan  added annotations to genes annotated in the previous steps and additionally annotated 42 (5%) male-enriched and 31 (4%) female-enriched genes that were previously unannotated. Annotations were assigned to a total of 307 (40%) male-enriched and 373 (51%) female-enriched genes (Additional file 1).
Overrepresented GO terms are presented in Additional file 5. In total, 75 GO terms were significantly overrepresented in sex-enriched genes (FDR < 0.05). Of these GO terms, 16 (21%) were overrepresented in males and 59 (79%) in females. Female-enriched genes were represented by several GO classes such as nucleotide binding, nitrogen metabolism, cell cycle, cytoskeleton organization and organelles. In contrast, male-enriched genes were dominated by GO terms related to transporter and channel activity (Additional file 5).
Identification and annotation of sex-enriched nutrient amino acid transporters in the aphid Myzus persicae
From the sex-enriched genes identified in the microarray, we identified five nutrient amino acid transporters overexpressed 4.2-fold to 20.1-fold in males (FDR < 0.05; Table 1 and Additional file 3). Four of the five male-enriched transporters were annotated in Blast2GO based on GO terms associated with significant BLAST hits (E < 0.001) and/or InterProScan IDs (Table 1 Additional file 3). The fifth transporter had no BLAST hits associated with GO terms, but its one InterProScan ID was annotated as belonging to transmembrane amino acid transporters (Table 1). After translating unigene nucleotide sequences to amino acid sequences based on the largest open reading frame, four of the five transporters significantly matched Pfam domains AA_permease (PF000324) or Aa_trans (PF01490) (Table 1).
Two transporters (contigs 1492 and 8321; See Additional file 4 for the GenBank accession numbers) were overexpressed 5.7 to 20.1-fold in males relative to both sexual and asexual females (Table 1 Additional file 3). We further annotated these two transporters based on their strongly male-biased expression. Using BLASTX and reciprocal TBLASTN searches, M. persicae contig 1492 (Table 1 Additional file 4) and the A. pisum amino acid permease ACYPI005156 were reciprocal BLAST best hits. These two genes also paired with unambiguous support in a gene phylogeny for insect orthologs to the Drosophilia gene slimfast, a cationic amino acid permease  of which A. pisum has a ten paralog aphid-specific expansion  (Figure 1). A BLASTX search for the other M. persicae contig, 8321 (Table 1 and Additional file 4), against the A. pisum nr protein database returned the A. pisum gene ACYPI003240 as the best hit, but the reciprocal TBLASTN search failed to return contig 8321 as the best hit for ACYPI003240. Phylogenetic analysis paired contig 8321 and ACYPI003240 with unambiguous support in the slimfast gene phylogeny (Figure 1). Based on the homology of the aphid expansion to slimfast, we named the 10 A. pisum paralogs ACYPIslif1-10 (Table 2).
Intrigued by having identified male-enriched nutrient amino acid transporters, we sought to discover if more sex-enriched amino acid transporters were represented in the microarray that our annotation pipeline had failed to annotate. TBLASTX and BLASTN searches queried all 40 A. pisum nutrient amino acid transporters [as identified in 9] against the M. persicae unigenes in the microarray. Apart from the 5 male-enriched transporters annotated above, we uncovered 9 additional contigs with significant (E < 0.001) similarity to A. pisum nutrient amino acid transporters. Of these 9 contigs, 5 were significantly (FDR < 0.05) male-enriched (two of which were male-biased) and 3 were female-enriched. Fold change for most of these contigs was less than four, explaining why we did not identify them in our initial analysis. In total, the M. persicae microarray targeted 14 contigs that significantly matched nutrient amino acid transporters, of which 10 were male-enriched or male-biased and 3 were female-enriched.
Sex-biased expression in the aphid slimfastexpansion
In our survey for more amino acid transporters in the microarray, we found no additional slimfast paralogs. We thus shifted the focus of our study to A. pisum so as to leverage its sequenced genome  to analyze the evolution of male-biased aphid slimfast. Expression was quantified by quantitative PCR (QPCR) between males and the three female morphs across three biological replicates. Expression was quantified for all slimfast paralogs but three, for which sequence similarity and AT richness in the untranslated regions prevented the design of paralog-specific primers .
Expression results are presented as a heat map aligned against the slimfast gene phylogeny in Figure 2. Paralogs ACYPIslif5, 6, and 10 were male-biased, with ACYPIslif5 and 6 showing the highest male-biased expression (Average fold change ± SD = 5.0 ± 2.2) and ACYPIslif10 showing less upregulation in males (Average fold change ± SD = 2.0 ± 0.5). Expression patterns for male-biased genes were consistent across all biological replicates (Figure 2). Additional patterns that were consistent across the three biological replicates were (1) enriched expression for ACYPIslif8 in asexual females at long day and (2) consistent low expression across all morphs for ACYPIslif9.
Molecular evolution of male-biased slimfastparalogs
The expression profiles of each slimfast paralog inspired the molecular evolutionary models we used to test if branches leading to male-biased paralogs experienced accelerated rates of evolution. Four codon-based branch models (depicted in Figure 3) [19, 20] were constructed to test for differences in the ratio of non-synonymous to synonymous substitution rates along specific branches (dN/dS = ω). The null model (one ratio) assumed equal ω for all branches. The first alternative model (two ratios) assumed different ω ratios for branches within and outside the aphid-specific slimfast expansion. The second alternative model (three ratios) assumed an additional, collective ω ratio for the branches leading to the three male-biased paralogs, and the third alternative model (five ratios) assumed a distinct ω for each branch leading to a male-biased paralog or clade (Figure 3).
Results for the models and likelihood ratio tests are given in Table 3. The two ratios model had a significantly higher likelihood than the one ratio model and predicted a higher ω for branches within the aphid slimfast expansion, consistent with previous work . Therefore, the remaining two models were compared to the two ratios model. The three ratios model was not significantly better than the two ratios model, but a significant improvement in likelihood was observed with the five ratios model. Using the five ratios model, branches leading to male-biased clades (ACYPIslif6, contig 1492 and ACYPIslif5, contig 8321) had lower ω values than the average branch within the expansion (0.17-0.18 vs. 0.22). In contrast, the branch leading to ACYPIslif10 had a greater ω value than other expansion branches (0.61 vs. 0.22).
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 slimfastexpansion
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 slimfastexpression 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.
This study is the first to report evidence for a male function of the nutrient amino acid transporter slimfast. Using a microarray and QPCR, we identified three male-biased slimfast paralogs in an aphid-specific gene family expansion, and two of those paralogs had conserved expression profiles across two aphid species. By integrating different sources of knowledge on the function and expression of slimfast and related nutrient amino acid transporters, we propose competing hypotheses for the function of male-biased slimfast in male-dependent nutritional, sensory, and reproductive functions.
This study highlights the necessity of examining different types of expression data for functionally annotating genes in novel genomes. While our previous work showed evidence that the aphid slimfast expansion is maintained in the A. pisum genome because some paralogs evolved a novel role in mediating symbiotic interactions , this study extends the hypothesis for the maintenance of the slimfast expansion to include paralog divergence for unexpected sex-dependent functions. The integration of different expression profiles to assist in functionally characterizing genes is particularly important for novel genes in all organisms, including gene duplications, which have the potential to evolve divergent functions from homologous genes present in single copies in other organisms.
Design, execution, and statistical analysis of microarray experiments
To investigate gene expression based on sex, Agilent microarrays targeting 10,478 M. persicae unigenes  and ESTs were used to quantify mRNA expression. The microarray measured gene expression between four treatments and two aphid lines (W109 and W115), both collected in 2008 on tobacco in Windsor, CT by ACCW and colleague. The four treatments were males and three different female reproductive morphs: sexual females, asexual females at short day, and asexual females at long day. We collected asexual females at long day from our standard asexual cultures (14:10 Light:Dark, 20°C). To induce sexual morphs, we simulated seasonal differences by changing the growth temperature and light/dark cycle (10:14 L:D, 16°C). When sexual morphs appeared, males, sexual females and asexual females at short day were collected.
Total RNA was extracted from ten flash frozen aphids per aphid line per treatment using an RNeasy Mini Kit (Qiagen, Valencia, CA). RNA quantity and quality was assessed using an RNA 6000 Chip on the Agilent 2100 Bioanalyzer. One microgram of total RNA was labeled using an Amino Allyl MessageAmp aRNA Amplification Kit (Ambion) and Cy3- and Cy5-NHS ethers (Amersham). Following labeling, the concentration of each sample was determined spectrophotometrically and equal amounts of labeled amplified RNA were competitively hybridized to the arrays. Factorial design was used for this microarray experiment. Four competitive hybridizations using the aphid line W109 (first biological replicate) were made as following: male → asexual female (long day) → sexual female → asexual female (short day) → male. The direction of the arrow indicates Cy3 → Cy5 sample labeling. An additional four competitive hybridizations were made with the reverse labeling (Cy5 → Cy3) using the aphid line W115 (second biological replicate).
Following hybridization and washing, microarrays were scanned at 5 μm resolution using a GenePix 4000B scanner (Molecular Devices). The resulting images were analyzed using GenePix Pro 6.1 (Molecular Devices) and subject to quality control using Acuity 4.0 (Molecular Devices). Data were analyzed using Linear Models for Microarray Data (LIMMA) implemented in R . Data normalization was executed first within each array using loess normalization, then normalization between arrays was executed using the LIMMA quantile algorithm. Differential expression and false discovery rates (FDR) were assessed using a linear model and empirical Bayes moderated F statistics [42, 43]. All primary microarray data are accessible through the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) database (http://www.ncbi.nih.gov/geo) under the GEO series accession number GSE31024.
Annotating sex-enriched genes
Sex-enriched genes identified in the microarray were annotated in Blast2GO v. 12.7.0 . Blast2GO implements a BLAST search and maps Gene Ontology (GO) annotations to the query sequences based on the best BLAST hits. Next, the annotation step evaluates candidate GO terms based on their hit similarity and annotation evidence codes (e.g. experimentally validated, computationally validated, etc.) to assign the most specific GO annotations possible to the original query sequences. The Blast2GO platform can also be used to perform an InterProScan to annotate sequences based on protein domain and motif signatures. GO terms corresponding to the InterPro annotation are then merged with the GO annotations assigned by the annotation step.
Sex-enriched M. persicae sequences were input as the query sequences and compared to the NCBI protein sequence database using a TBLASTN search, keeping up to 50 top hits at an e-value of 0.001 or less. Annotation was performed in two rounds to maximize both the quality and number of annotations. First, the annotation step was run with default evidence code weights, except that the evidence code IEA (Inferred from Electronic Annotation) was weighted 0. For the second round, unannotated sequences were passed through the annotation step using default evidence code weights. After the annotation step, we ran the InterProScan and merged the InterPro results with the previous annotations. Annotations were then augmented using Annex, a database of manually reviewed relationships between the three GO categories . Finally, the GO-slim function was used to summarize the annotation results. A fisher exact test was implemented in Blast2GO to examine overrepresented GO terms in male-enriched and female-enriched genes. Significance was assessed by a FDR of 0.05 or less.
Identification and annotation of male-enriched amino acid transporters
Male-enriched amino acid transporters were annotated based on GO terms and InterProScan IDs from a list of sex-biased genes found in the microarray. Annotations were manually validated by searching for protein domains in Pfam by hidden markov models (http://pfam.sanger.ac.uk/). Two male-biased amino acid transporters were assigned orthology to genes in the pea aphid, A. pisum, by reciprocal BLAST and phylogenetic analysis. Briefly, the M. persicae sequences were queried against the A. pisum non-redundant (nr) protein database on NCBI using a BLASTX search. Query sequences were contigs assembled from M. persicae ESTs  (available at http://www.aphidbase.com/aphidbase/downloads). The top A. pisum BLAST hit for each query was subsequently subjected to a local TBLASTN search against the entire set of M. persicae unigenes in the microarray.
For the phylogenetic analysis, male-biased M. persicae contigs were translated to protein and aligned to a profile of A. pisum homologs in SeaView v. 4  using a Clustal W  plug-in. The profile, from Price et al., , consisted of all A. pisum members of the slimfast gene family, slimfast sequences from select other insects, and two outgroup sequences closely related to the slimfast gene family . The new alignment was then used to build a phylogeny in MrBayes v. 3.1.2  using two simultaneous runs, each with four chains. Chains were run for two million generations, after which the standard deviation of split frequencies between the runs was less than 0.01. Convergence was assessed in Tracer v. 1.5 . Briefly, Tracer plots the value of the log-likelihood and model parameters against the number of generations, visually displaying parameter convergence between the two runs and enabling us to determine that MrBayes sufficiently sampled the parameter space. A consensus tree was constructed after discarding generations making up the burn-in, as determined in Tracer v. 1.5 .
Analyzing sex-biased gene expression of slimfast homologs in A. pisum
Sex-dependent expression for seven out of ten A. pisum slimfast homologs was quantified by performing QPCR on three different A. pisum clonal lineages that included two lineages with intermediate lifecycles, LSR1  and 7A  and one holocyclic lineage, 9-2-1  (see  for a description of the morphs produced by different aphid life cycle classes). The three paralogs for which we did not quantify expression shared so much sequence similarity and untranslated regions were so AT rich (~80%) that we were unable to design paralog-specific primers. For the two intermediate lineages (LSR1 and 7A), QPCR was performed on the same aphid morphs used in the microarray, asexual females at long day, asexual females at short day, sexual females, and males. For the holocyclic lineage (9-2-1), which completely switches to the production of sexual morphs at short day, QPCR was performed on asexual females at long day, sexual females, and males. Males and female morphs were collected as described above.
Total RNA was extracted from at least 6 whole, wingless adults of each morph using an RNeasy mini kit (Qiagen), and a DNase digest was performed to eliminate genomic DNA. cDNA was synthesized from 450 ng of total RNA using qScript cDNA SuperMix (Quanta Biosciences) and QPCR was performed using PerfeCTa® SYBR® Green FastMix® (Quanta Biosciences). Gene expression was compared between morphs using 2-ΔΔCTmethodology , with expression between morphs normalized to the expression of housekeeping gene glyceraldehyde-3-dehydrogenase (GAPDH, ACYPI009769 ). Experiments were each performed in triplicate and included no template controls. Prior to running experiments, we verified that cDNA pools lacked genomic DNA by running no reverse transcription controls alongside no template controls and positive controls. QPCR reactions were performed on a Mastercycler® ep realplex4 real-time PCR system (Eppendorf) using a program that began with 95°C for 5 minutes, followed by 40 cycles at 95°C for 15 sec, 52°C for 15 sec, and 60°C for 20 sec. Amplification profiles and melt-curves were analyzed in Mastercycler® ep realplex software v. 1.5 (Eppendorf). For information on primer sequences and efficiencies, see . Gene expression data were collectively normalized, allowing comparison of expression profiles across paralogs and aphid lines. Normalization was performed by converting ΔCT values to z-scores as follows:
The normalized expression for each gene from the three lines was compiled into a heat map where yellow indicates z > 0 and blue indicates z < 0.
Molecular evolution analyses
Rates of molecular evolution along phylogenetic tree branches were estimated for branches in the aphid specific slimfast expansion. The analysis was conducted using the same phylogeneny for which the methods were described above. The amino acid alignment used to reconstruct the tree was converted back to a codon alignment in SeaView v. 4 , and the codon alignment was used to estimate the ratio of non-synonymous (dN) to synonymous (dS) substitution rates along branches, or dN/dS (also denoted ω). The ω ratio reflects the strength and type of selection in protein coding sequences. Excess dS (ω < 1) indicates that selection favors substitutions that conserve the amino acid sequence (purifying selection), while equal dS and dN (ω = 1) illustrates relaxed selective constraints (neutral selection). Excess dN (ω > 1) indicates that selection favors substitutions that change the amino acid sequence (positive selection).
The ω ratio was estimated for different branches in the aphid slimfast expansion by maximum likelihood in the PAML package . Analyses implemented the branch models [19, 20] with one or more ω categories for branches and one ω across sites (model = 0 or 2, NSsites = 0). The parameters ω and κ (transition rate/transversion rate) were both estimated, starting from initial values of 0.2 and 2 respectively, and codon frequency was measured using a 3 × 4 codon table. We tested for elevated ω along specific branches using various models. Briefly, we tested the null model assuming equal ω across branches and several nested models allowing for different ω ratios for particular groups of branches and compared the log likelihoods under these models with a likelihood ratio test [LRT, ]. If the LRT showed a statistically significant improvement in log likelihood from one model to the next, then the model with the higher log likelihood was supported. Models we tested were inspired by the expression profiles of aphid paralogs in the expansion and we outline them in the results section.
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We thank W. Huang for helping to collect M. persicae lines used for the microarray experiment and T. Wyss and S. Echnadia for technical assistance. We also thank D. Crawford, B. Browne, D. Hedges, D. R. G. Price and two anonymous reviewers for providing helpful discussion and comments. This work was supported by USDA grant 2005-35604-15446 and University of Miami Start-up funds to ACCW.
ACCW designed the microarray experiment. LN performed the image and statistical analysis on the microarray data. RPD identified and annotated sex-enriched genes from the microarray. ACCW and RPD conceived and designed the QPCR experiments. RPD performed the QPCR experiment. RPD conceived, designed and executed the molecular evolution analyses. RPD and ACCW wrote the paper. All authors have read and approved the final manuscript.