Sex determination in insects is characterized by a gene cascade that is conserved at the bottom but contains diverse primary signals at the top. The bottom master switch gene doublesex is found in all insects. Its upstream regulator transformer is present in the orders Hymenoptera, Coleoptera and Diptera, but has thus far not been found in Lepidoptera and in the basal lineages of Diptera. transformer is presumed to be ancestral to the holometabolous insects based on its shared domains and conserved features of autoregulation and sex-specific splicing. We interpret that its absence in basal lineages of Diptera and its order-specific conserved domains indicate multiple independent losses or recruitments into the sex determination cascade. Duplications of transformer are found in derived families within the Hymenoptera, characterized by their complementary sex determination mechanism. As duplications are not found in any other insect order, they appear linked to the haplodiploid reproduction of the Hymenoptera. Further phylogenetic analyses combined with functional studies are needed to understand the evolutionary history of the transformer gene among insects.

The development into different sexes is a widespread phenomenon in eukaryote organisms. Despite its fundamentality, the mechanisms governing the determination and development of the sexes are widely diverged, even on small evolutionary time scales and between related organismal groups [Bull, 1983; Marshall Graves, 2008]. We are still far from grasping the full breadth of this diversity, and we have very little understanding of the evolutionary forces that drive it. Only a limited number of animal taxa have been investigated to a considerable extent. Sex determination in mammals and birds is rather invariable, but notably fish and insects are known for their rapid turnovers in the function and nature of sex determination genes [Devlin and Nagahama, 2002; Sanchez, 2008]. In this short review, we consider the genetic regulation of sex determination in insects with a focus on the phylogenetic distribution of 2 important genes, doublesex and transformer. Sex determination in insects is characterized by a gene cascade that is conserved at the bottom but contains diverse primary signals at the top. The bottom master switch gene is doublesex and is found in all insects studied to date. Its upstream regulator is transformer whose phylogenetic distribution shows some remarkable patterns, being present in some groups but not in others (see below).

Sex determination in insects is organized as a hierarchical order of genes which is termed a genetic cascade. A primary signal is passed through a series of sex determination genes ending with a downstream master switch gene which regulates sexual differentiation genes. The downstream genes are more conserved than the upstream ones, which is explained by the cascade evolving from the bottom upwards [Wilkins, 1995]. The master switch at the bottom of the sex determination cascade is identified as doublesex in a range of insect species, including Diptera (flies and mosquitoes), Hymenoptera (ants, bees, wasps, and sawflies) and Lepidoptera (butterflies and moths) [Shukla and Nagaraju, 2010a]. doublesex belongs to a family of DM-domain genes which appear to play a role in sex determination or sexual differentiation in all Metazoa [Suzuki et al., 2001; Hodgkin, 2002; Kopp, 2012]. It is a transcription factor that regulates downstream sexual differentiation genes [Shukla and Nagaraju, 2010a]. doublesex is alternatively spliced by transformer into sex-specific variants, which leads to the production of male- and female-specific DSX proteins.

Genes upstream of doublesex are less conserved in the sex determination cascade. The gene directly upstream of doublesex is transformer which was considered a constant feature in insects, but this idea is now challenged (see below). The transformer gene is not functionally conserved outside of insects. It is for example present in the water flea Daphnia magna, but not involved in the environmental sex determination of this crustacean [Kato et al., 2010]. Transformer belongs to a class of SR-type proteins that have sequences containing high frequencies of arginine (R) and serine (S) residues (fig. 1a). It contains several conserved domains, one of which is only shared amongst the Diptera (‘the dipteran domain'), and another only present in the Hymenoptera (‘the hymenopteran domain'). The function of these domains remains unknown. transformer is a very rapidly evolving gene and regulates fruitlesssplicing as well [Ryner et al., 1996; Gailey et al., 2006], in addition to doublesex. Sex-specific transformer mRNA is produced by alternative splicing, a regulatory mechanism that is sustained by an autoregulatory loop. When female transformer transcripts are blocked by e.g. RNA interference, it results in male development, as shown for several dipterans [Pane et al., 2002; Lagos et al., 2007] and the hymenopterans Apis mellifera [Hasselmann et al., 2008] and Nasonia vitripennis [Verhulst et al., 2010a].

Fig. 1

The domain structure of transformer (a) and doublesex(b) in the various holometabolous insect orders. a The transformer order-specific domains of Hymenoptera (HYM) and Diptera (DIP) are shown in light grey. The autoregulation domain (CAM) is found in all examined species except Drosophila. The arginine- and serine-rich region (Arg/Ser) and the proline-rich region (Pro-rich) are present in all orders. b The DM-domain and the dimerization domain (Dimer) of doublesexare shown.

Fig. 1

The domain structure of transformer (a) and doublesex(b) in the various holometabolous insect orders. a The transformer order-specific domains of Hymenoptera (HYM) and Diptera (DIP) are shown in light grey. The autoregulation domain (CAM) is found in all examined species except Drosophila. The arginine- and serine-rich region (Arg/Ser) and the proline-rich region (Pro-rich) are present in all orders. b The DM-domain and the dimerization domain (Dimer) of doublesexare shown.

Close modal

The primary signals on top of the sex determination cascade vary strongly among insects. In Drosophila melanogaster, the X chromosome:autosome ratio functions as the primary signal. The concentration of X-linked signal elements activates the Sex-lethal early promoter [Erickson and Quintero, 2007], which in turn regulates transformersplicing. In other dipterans, Sex-lethal is not an intermediary between the primary signal and transformer, but transformer is directly regulated by the absence or presence of a male-determining M factor [Dübendorfer et al., 2002; Pane et al., 2002; Hediger et al., 2010]. The M factor has not been identified in any insect yet. In haplodiploid Hymenoptera, the primary signal is the ploidy level of the egg (diploid = female, haploid = male), but how this signal is transmitted towards transformer is still subject of active research [Verhulst et al., 2013; Verhulst and van de Zande, this issue]. In Lepidoptera, which have female heterogamety, a presumably dominant female determiner on the W chromosome regulates a splicing inhibitor (PSI) of doublesex [Suzuki et al., 2008]. The number of investigated insect species is still small, and given their enormous variety in genetic systems, it is to be expected that many more regulatory mechanisms of sex determination will be discovered in the future.

The downstream master switch gene doublesex was first identified in D. melanogaster [Baker and Wolfner, 1988]. The protein is characterized by 2 functional domains: a DNA-binding domain (DM or OD1) and a dimerization domain (OD2) (fig. 1b). The DM domain is found in multiple genes of the DM superfamily group consisting of doublesex, mab3 and related transcription factors (Dmrt), while the dimerization domain is exclusively found in doublesex. Orthologs of doublesex were found in each insect species examined thus far (references in table 1), covering a range of Diptera, Lepidoptera, Coleoptera, and Hymenoptera. All of these belong to the derived group of holometabolous insects, while no information has been published from more basal insects. Other DM superfamily genes outside of the insects do not contain a dimerization domain and do not exhibit the sex-specific splicing typical of doublesex.

Table 1

Presence of doublesex, transformer, transformer paralogs and the transformer autoregulation domain (CAM) in insect species. Species with a genomic database from which transformer was characterized were not assessed for presence of doublesex if the family belonged to a well-studied order (Diptera and Hymenoptera)

Presence of doublesex, transformer, transformer paralogs and the transformer autoregulation domain (CAM) in insect species. Species with a genomic database from which transformer was characterized were not assessed for presence of doublesex if the family belonged to a well-studied order (Diptera and Hymenoptera)
Presence of doublesex, transformer, transformer paralogs and the transformer autoregulation domain (CAM) in insect species. Species with a genomic database from which transformer was characterized were not assessed for presence of doublesex if the family belonged to a well-studied order (Diptera and Hymenoptera)

The increasing public availability of genomic and transcriptomic databases gave us the opportunity to screen more insect species for the presence of doublesex. We used the presence of the dimerization domain in combination with an upstream DM domain as evidence for the presence and functional conservation of doublesex (see Appendix). We found indications for putative orthologs of doublesex in a number of primitive insect groups (table 1; online supplementary table 1, see www.karger.com/doi/10.1159/000357056). The genome of Pediculus humanus corporis (human body louse) contains a hypothetical protein (XM_002427625.1) which is a candidate for doublesex, consisting of both a DM domain and a putative dimerization domain. A homolog of the dimerization domain could, however, not be found in 2 hemipteran genomes, Acyrthosiphon pisum and Rhodnius prolixus, whereas both species do contain 2 DM superfamily genes. The closest relative to the insects examined thus far is the water flea D. magna which is claimed to have 2 doublesexgenes, neither of which shows the sex-specific splicing characteristic of insects, but both do contain a putative dimerization domain [Kato et al., 2011]. It thus appears that the sex-specific splicing feature of doublesex was acquired early in the evolution of insects. More species from primitive groups such as Palaeoptera, Zygentoma and Archaeognatha need to be investigated to assert its precise origin. Further, identification of doublesex orthologs in genomic assemblies provides no evidence for sex-specific splicing and its coincidence with the origin of a conserved dimerization domain. Functional analysis outside the holometabolous insects is needed to ascertain its appearance in the sex determination cascade and the conservation of this role in various groups.

Within the Holometabola, doublesex appears to be ubiquitously present, with possible identifications from this study in genomes of Coleoptera (Dendroctonus ponderosae) and Diptera (Mayetiola destructor and Phlebotomus papatasi) (table 1; online suppl. table 1). We could also find it with both of its conserved functional domains in the order of Strepsiptera (Mengenilla moldrzyki).

transformer, the upstream splicing controller of doublesex, shows a distinctly more patchy distribution among insects (fig. 2). It is also more diverged in sequence than doublesex, which complicates its identification in genome sequences. The most conserved part of transformer is the autoregulation domain CAM (from Ceratitis capitata-Apis mellifera-Musca domestica; nomenclature based on Hediger et al. [2010]). This domain is present in all transformer genes, except in that of D. melanogaster. In Drosophila, Sex-lethal has been added upstream of transformer, and it has taken over autoregulation, making the CAM domain of transformer obsolete [Bopp, 2010]. Other features of transformer include order-specific domains of unknown function. One of these is shared among all Hymenoptera, the other is present in all Diptera (fig. 1a). In addition, 2 regions are present in all transformers: an arginine/serine-rich region and a proline-rich region. The location of these regions towards the C-terminal is conserved across all transformer-containing insect species studied to date, while the location of the CAM domain and the order-specific domains varies. In Hymenoptera, the order-specific domain is located upstream of the CAM domain, whereas in Diptera, its order-specific domain is close to the arginine/serine region. In non-drosophilid Diptera, the CAM domain is positioned towards the N-terminal of the gene, a feature that it shares with the short transformer gene in the beetle Tribolium castaneum.

Fig. 2

Phylogeny of insects showing the presence of transformer(tra) across orders. Orders are noted on the right and superorders are marked in capital letters. Proposed gain of transformer into the sex determination cascade is indicated by the grey box, with inserted white boxes indicating secondary losses. Question marks refer to uncertainties as data are based only on this study and require validation. The Diptera are in a light grey box to mark the variable presence of transformer in this order, which is characterized by an absence in basal lineages and a presence in derived lineages, with the exception of Drosophila. The addition of the Hymenoptera and Diptera domains in transformer is marked by boxes labeled HYM and DIP. The phylogeny is based on Trautwein et al. [2012].

Fig. 2

Phylogeny of insects showing the presence of transformer(tra) across orders. Orders are noted on the right and superorders are marked in capital letters. Proposed gain of transformer into the sex determination cascade is indicated by the grey box, with inserted white boxes indicating secondary losses. Question marks refer to uncertainties as data are based only on this study and require validation. The Diptera are in a light grey box to mark the variable presence of transformer in this order, which is characterized by an absence in basal lineages and a presence in derived lineages, with the exception of Drosophila. The addition of the Hymenoptera and Diptera domains in transformer is marked by boxes labeled HYM and DIP. The phylogeny is based on Trautwein et al. [2012].

Close modal

Hymenoptera are the most basal order of the holometabolous insects (fig. 2). transformerhas been identified in more than a dozen hymenopteran species, all belonging to the more derived lineages within the order. On the other hand, in Lepidoptera no transformer homologs have been found thus far. Most information is available for the well-studied species Bombyx mori [Suzuki, 2010], but genomic data of Danaus plexippus and Heliconius melpomene indicate lack of a transformer homolog as well (table 1; Appendix). With only 1 published Strepsiptera genome, that of Mengenilla moldrzyki[Niehuis et al., 2012], information about this group is scarce, but this species appears to lack a transformer homolog. This is remarkable because as a sister order of the Coleoptera, it was presumed to carry transformer, based on its presence in T. castaneum [Shukla and Palli, 2012a]. However, in the genome of the coleopteran D. ponderosae, which was published recently [Keeling et al., 2013], we could also not find a homolog of transformer. This leaves the question of whether transformer is conserved in Coleoptera open until more species are examined.

An interesting pattern of transformer distribution emerges in the Diptera, where recent studies have suggested it to be absent in mosquitoes, which form a basal dipteran lineage [Salvemini et al., 2013]. transformer is found in all more derived Brachycera species (references in table 1) including D. melanogaster, where it has lost its CAM domain (fig. 3). In addition to its apparent absence in several mosquito species, we could not find transformer in M. destructor (table 1; Appendix), which belongs to the basal lineage of the Bibionomorpha, in Lutzomyia longipalpis (Psychodomorpha) and, peculiarly, in Megaselia scalaris (a derived brachyceran species). On the other hand, the recently published genomic assembly of P. papatasi, a member of the basal Psychodomorpha, contains a putative transformer homolog, but misses the CAM domain. A cautionary note is needed here, as this absence of its most reliable recognition feature complicates its identification (see Appendix). The putative P. papatasi homolog does contain a Diptera domain and structurally resembles the CAM domain-lacking D. melanogaster transformer. Outside of the Hymenoptera, no paralogs of transformerhave been identified, thus indicating that this putative homolog in P. papatasi has a likely orthologous origin. Interestingly, the CAM domains of the more derived Diptera show strong similarity with the more than a dozen known sequences of the Hymenoptera and the coleopteran Tribolium. The D. magnatransformer also contains a highly conserved CAM domain, but it is located downstream of the arginine/serine-rich region and is not involved in sex determination [Kato et al., 2010]. This suggests that other selective pressures have acted to preserve its presence; an explanation for this could be that the domain serves additional functions in development.

Fig. 3

Phylogeny of dipteran insects showing the distribution and structure of transformer (tra). The figure is based on phylogenetic trees by Bertone et al. [2008], Lambkin et al. [2013] and Wiegmann et al. [2011]. There is no consensus about the resolution of the basal lineages.

Fig. 3

Phylogeny of dipteran insects showing the distribution and structure of transformer (tra). The figure is based on phylogenetic trees by Bertone et al. [2008], Lambkin et al. [2013] and Wiegmann et al. [2011]. There is no consensus about the resolution of the basal lineages.

Close modal

The presence of transformer in Daphnia indicates that it arose before the branching of Crustacea and Hexapoda. Under the assumption of a unique transformerrecruitment into the sex determination cascade at the basis of the holometabolous insects, secondary loss of transformer must have occurred on at least 2 occasions based on current studies: in the Lepidoptera [Mita et al., 2004] and the basal Diptera [Salvemini et al., 2013]. We found evidence for 2 additional possible losses, i.e. in a strepsipteran and a coleopteran species. Secondary loss of the CAM domain was only known from D. melanogaster, but our study suggests a second dipteran case, i.e. the sand fly P. papatasi. The absence of transformer in groups other than holometabolous insects and crustaceans suggests that the gene can disappear again. This is at odds with the presumed conserved role in sex determination, but may reflect that transformer was only introduced in the sex determination cascade at the rise of the holometabolous insects.

The current knowledge on the distribution and structural features of transformer is based on a small collection of insect species. Studies have only been performed on 4 orders (Coleoptera, Diptera, Hymenoptera, and Lepidoptera) out of the 12 that make up the holometabolous insects. This review includes a first indication of a lack of transformerin a fifth order (the Strepsiptera), but no attention has been given to the remaining 7 orders, due in part to lack of genomic/transcriptomic data. Non-holometabolous insects comprise another 17 orders, amongst which no species have been studied regarding their sex determination (genes).

For a long time, D. melanogaster was the only species known to have an additional regulator upstream of transformer in the sex determination cascade, i.e. Sex-lethal. A few years ago, Beye et al. [2003] and Hasselmann et al. [2008a] found that the honey bee (A. mellifera) had another gene inserted at the top of its cascade. This turned out to be a duplication of transformer(=feminizer in A. mellifera).The duplication initiates the female-specific splicing of feminizer transcripts. The zygosity of this complementary sex determiner (csd) locus determines the switching on of the autoregulation loop of transformer/feminizer [Beye et al., 2003; Hasselmann et al., 2008a]. Feminizer genes coupled with paralogous csdgenes were also found in 2 sister species, Apis cerana and Apis dorsata [Hasselmann et al., 2008b]. The 3 species showed signs of convergent evolution based on sharing of the same nonsynonymous substitutions in different alleles across species. The Apis genus belongs to the Aculeata, consisting of bees, vespoid wasps and ants, which is considered a derived lineage within the Hymenoptera. Two recent reports [Schmieder et al., 2012; Privman et al., 2013] found a similar duplication of transformer in 2 bumblebees and 6 out of 7 sequenced ant species (listed in table 1), all belonging to the Aculeata. The presence of transformer paralogs in these aculeate species is explained by a single duplication event after which the genes evolved through concerted evolution [Schmieder et al., 2012; Privman et al., 2013]. This places the duplication of transformer/feminizer leading to the csd gene before the appearance of the Aculeata lineages, around 120 million years ago [Schmieder et al., 2012]. In 3 examined cases, the transformer/feminizer genes show sex-specific splicing, while the duplicated transformer genes are expressed but without a sex-specific splicing pattern [Schmieder et al., 2012]. No functional analysis has been performed on these paralogs of transformer, thus leaving open the question whether they function similar to the csd gene and are part of the sex determination cascade.

Duplications of transformer have thus far only been identified in Hymenoptera, being haplodiploid, and exclusively in species presumed to have a complementary sex-determining mechanism (CSD). Under CSD, individuals homozygous at the csd locus develop into males, while heterozygosity at the csd locus starts female development [Whiting, 1943; Beye et al., 2003]. To counteract the costs of inbreeding, where decreasing variation would result in diploid homozygous males, the csd gene shows high allelic variation [Hasselmann et al., 2008b]. This effect of inbreeding, which results in diploid males, often sterile or inviable, allows simple testing for presence of CSD, at least in species that can be cultured in the laboratory. CSD has been inferred in over 60 species [van Wilgenburg et al., 2006] and is presumed to be ancestral to the Hymenoptera [Cook, 1993; Heimpel and de Boer, 2008; Asplen et al., 2009]. Most studies indicated CSD based on a single locus (sl-CSD), but studies in the Cotesiagenus have found evidence for multiple loci (multi-locus, or ml-CSD) [de Boer et al., 2008, 2012].

Based on the widespread occurrence of both transformer paralogs and CSD within the Aculeata, it is tempting to propose that the CSD locus consists of the transformer paralog in this group, as documented for the honey bee [Beye et al., 2003; Hasselmann et al., 2008a]. The paralog would then need to contain a hypervariable region as in the A. melliferacsdgene, or at least a high variety of alleles. However, within the Aculeata, 2 of the thus far examined species appear to lack a duplication of transformer: the leaf-cutting ant Acromyrmex echinatior [Nygaard et al., 2011; Schmieder et al., 2012; Privman et al., 2013], which is known to produce high levels of diploid males, and the solitary bee Megachile rotundata (table 1). No duplications have yet been found outside the Aculeata, but the availability of databases in Hymenoptera is strongly skewed towards eusocial derived species. Unfortunately, of the many other species that have CSD [van Wilgenburg et al., 2006], no genomic or transcriptomic information is available. Data on lineages of sawflies, which are basal to the Hymenoptera that are in turn one of the basal orders of the holometabolous insects, is crucial for determining whether transformer is involved in the presumed CSD ancestry. In the chalcidoid N. vitripennis,csd is absent, and there are no duplications of transformer. Its sex determination is based on a maternal effect genomic imprinting model [Verhulst et al., 2010a, 2013; Verhulst and van de Zande, this issue]. N. vitripennis is the first species outside the Aculeata that has no CSD and for which genomic data is available. More studies on both non-CSD and non-Aculeata species are needed to assess the commonality of the csd/transformer mechanism and the abundance of maternal imprinting sex determination among Hymenoptera.

In this article, we considered the phylogenetic distribution and evolutionary dynamics of 2 important sex determination genes, doublesex and transformer, among the insects. Although members of only a limited number of orders have been investigated, it is safe to conclude that doublesex serves as the master switch at the bottom of the sex determination cascade in all insects. The gene and its characteristic domains, as well as its sex-specific splicing feature, are found across holometabolous insects. Branches outside of the Holometabola, i.e. P. humanus corporis in the Phthiraptera, contain homologous genes which show less conservation in the insect-specific dimerization domain. This latter feature cannot be identified when moving further into the basal lineages.

transformer, the upstream regulator of doublesex, has a less ubiquitous distribution. It is found outside holometabolous insects, but there it apparently has no function in sex determination, e.g. in D. magna [Kato et al., 2010]. It is apparently (partly) absent in 4 insect orders, notably the Lepidoptera, basal Diptera, Strepsiptera, and Coleoptera. transformer could have been recruited multiple times into the sex determination cascade, but its domain structure (CAM domain followed by an arginine/serine domain and a proline-rich domain) is conserved across all groups. An origin of a sex determination function at the basis of the holometabolous insects appears most parsimonious, but requires multiple independent losses. If transformer, however, originated before the branching-off of the crustaceans, it must have been lost in more groups outside of insects as well.

Many additional questions remain to be answered about transformer before we can make further inferences about its evolution in insects. First of all, a broader taxonomic screen is needed to determine how widespread transformer is present among insects. Particularly informative groups are the Paraneoptera (lice, thrips and bugs), a sister group of the Holometabola, and some of the other basal taxa, such as the Odonata (damselflies and dragonflies) and Ephemeroptera (mayflies). These could provide answers to the precise timing of transformer recruitment into the insect sex determination pathway. No information is available outside of the Holometabola, e.g. the Polyneoptera that span more than 10 orders of insects.

The evolutionary significance of transformer duplication in the Hymenoptera remains an enigma. Study of the basal lineages, such as the solitary Symphyta (sawflies), will be informative, for example to determine whether a link exists between transformer duplications and CSD. Only in the honey bee, the duplicated copy has been shown to have a functional role in sex determination. No functional studies have been performed on any other species with a transformer paralog. We also need more information about possible additional roles of transformer in development, as this may be revealing about the function of order-specific domains and may also yield insights into why some regions, such as the CAM domain, are conserved over a broad range of arthropod taxa. Our analysis found further support for Wilkins [1995] hypothesis that the genetic pathway of sex determination started with the most downstream signal and evolved by adding genes upstream. We also adhere to the argument of Verhulst et al. [2010b] that transformer plays a central role in the evolving cascade in some insect groups. However, its absence in some groups also shows that transformer can be circumvented, and alternative regulators of doublesex can evolve. The frequent gains and losses of genes and gene domains are another signifying feature of the insect sex determination cascade.

Next generation sequencing is exponentially increasing the amount of data across an increasing number of arthropod groups. Large sequencing projects on hundreds of arthropod species, in particular insects, are currently underway. The i5K project (http://arthropodgenomes.org/wiki/i5K), for example, plans to sequence 5,000 insect and other arthropod genomes in the next couple of years. The 1KITE project (http://www.1kite.org/) focuses exclusively on insects and will yield 1,000 insect transcriptomes spanning all orders. These efforts, combined with the increasing amount of separately published genomic and transcriptomic datasets, provide a great wealth of information for gene identification studies. Transcriptomic datasets can offer immediate tests whether the putative sex determination genes are expressed in the sequenced tissue and may even provide a first glimpse at sex-specific splice variants. Genomic databases have the advantage of structure detection and, if the scaffolds are large enough, synteny of the sex determination regions. In combination, they make the future for insect sex determination researchers a bright one.

Methodology for Identification of doublesex and transformer (Duplication) Homologs with tBLASTn Searches on Publicly Available Genomic Databases

The strong divergence of sex determination genes makes identification of homologs, particularly in thus far unexamined orders, not a straightforward task. Published studies which examined newly identified sex determination genes in genomic or transcriptomic databases relied on BLAST searches. Profile hidden Markov models could potentially be a more powerful method to detect distant homologs of these fast evolving genes.

The conserved domains provide some anchor points for the presence of a sex determination gene, though, on its own, this information cannot provide any evidence for an actual role in sex determination. Absence of a gene is more difficult to prove: is the gene truly lost (or never gained) in the species, or is it overlooked due to strong divergence and possible loss of conserved domains?

To increase detection of sex determination genes in unstudied insect orders and other relative outgroups, we searched for the presence of doublesex and transformer in a range of publicly available databases. We had no opportunity for any verification tests regarding the structure, expression or function of these genes. Moreover, some of the investigated species are distantly related to any studied species with an identified transformer or doublesex homolog, which added further difficulty to the scans.

Identification of doublesex and transformer homologs was performed in genomic databases of Pediculus humanus corporis, Acyrthosiphon pisum, Rhodnius prolixus, Megachile rotundata, Danaus plexippus, Heliconius melpomene, Dendroctonus ponderosae, Mengenilla moldrzyki, Lutzomyia longipalpis, Mayetiola destructor, Megaselia scalaris, and Phlebotomus papatasi with translated BLAST [Altschul et al., 1997]. As queries, the full Doublesex and Transformer protein sequences of Apis mellifera (ABW99105, NP_001128300), Nasonia vitripennis (ACJ65507, NP_001128299), Tribolium castaneum (AFQ62106, AFQ62109), Ceratitis capitata (AAN63597, AAM88673), Drosophila melanogaster (NP_731197, NP_524114) and Daphnia magna (BAJ78307/BAJ78309, BAI66432) were used. In addition, the dimerization domain of doublesex and the CAM, Hymenoptera and Diptera domains of transformer were used, as these conserved regions allow more stringent detection where the full sequences may provide false positives.

We thank Louis van de Zande, Eveline Verhulst, Wen-Juan Ma and Daniel Bopp for helpful discussions on insect sex determination and an anonymous reviewer for constructive criticism. This research was supported by TOP grant no. ALW 854.10.001 of the Netherlands Organisation for Scientific Research.

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