Characterization of the doublesex gene within the Culex pipiens complex suggests regulatory plasticity at the base of the mosquito sex determination cascade
© Price et al. 2015
Received: 24 November 2014
Accepted: 22 May 2015
Published: 11 June 2015
The doublesex gene controls somatic sexual differentiation of many metazoan species, including the malaria mosquito Anopheles gambiae and the dengue and yellow fever vector Aedes aegypti (Diptera: Culicidae). As in other studied dipteran dsx homologs, the gene maintains functionality via evolutionarily conserved protein domains and sex-specific alternative splicing. The upstream factors that regulate splicing of dsx and the manner in which they do so however remain variable even among closely related organisms. As the induction of sex ratio biases is a central mode of action in many emerging molecular insecticides, it is imperative to elucidate as much of the sex determination pathway as possible in the mosquito disease vectors.
Here we report the full-length gene sequence of the doublesex gene in Culex quinquefasciatus (Cxqdsx) and its male and female-specific isoforms. Cxqdsx maintains characteristics possibly derived in the Culicinae and present in the Aedes aegypti dsx gene (Aeadsx) such as gain of exon 3b and the presence of Rbp1 cis-regulatory binding sites, and also retains presumably ancestral attributes present in Anopheles gambiae such as maintenance of a singular female-specific exon 5. Unlike in Aedes aegypti, we find no evidence for intron gain in the female transcript(s), yet recover a second female isoform generated via selection of an alternate splice donor. Utilizing next-gen sequence (NGS) data, we complete the Aeadsx gene model and identify a putative core promoter region in both Aeadsx and Cxqdsx. Also utilizing NGS data, we construct a full-length gene sequence for the dsx homolog of the northern house mosquito Culex pipiens form pipiens (Cxpipdsx). Analysis of peptide evolutionary rates between Cxqdsx and Cxpipdsx (both members of the Culex pipiens complex) shows the male-specific portion of the transcript to have evolved rapidly with respect to female-specific and common regions.
As in other studied insects, doublesex maintains sex-specific splicing and conserved doublesex/mab-3 domains in the mosquitoes Culex quinquefasciatus and Cx. pipiens. The cis-regulated splicing of Cxqdsx does not appear to follow either currently described mosquito model (for An. gambiae and Ae. aegypti); each of the three mosquito genera exhibit evidence of unique cis-regulatory mechanisms. The male-specific dsx terminus exhibits rapid peptide evolutionary rates, even among closely related sibling species.
The manifestation of distinct sexes is fundamentally conserved among most metazoans. However, the development of sex-specific somatic and gonadal tissues and neuronal processes (e.g. behaviors) is governed by a variety of factors both environmental and genetic, and often varying widely between and within taxa [1–3]. Most animals direct sex-specific cell fate by function of the Doublesex/Mab-3 Related Transcription factor (DMRT) family of zinc-finger proteins [4, 5] and the genes they regulate. Within the insects, this process involves a genetic cascade first elucidated in the model fly Drosophila melanogaster  whereby a primary signal triggers sex-specific splicing of one or more regulatory factors which subsequently bind pre-mRNA of the conserved DMRT “major switch” gene, doublesex, and direct its sex-specific splicing, thus initiating development of male or female forms .
Although there are many diverse primary signals that initiate the cascade (e.g. X:A ratio, M-factors, W/Y chromosomes; see ), dsx appears to be conserved as the major switch at the base of the cascade [8, 9]. In many insects the male and female-specific splicing of dsx is directed by the upstream regulator transformer, a serine/arginine rich (SR) protein which itself is transcribed in a sex-specific manner, as well as the constitutively expressed transformer-2 [10, 11]. The resultant TRA/TRA2 peptide complex binds the dsx mRNA at the dsx repeat element (dsxRE), facilitated by the purine-rich enhancer (PRE) element [12, 13], and directs sex-specific splicing of dsx mRNA for translation into male (DSXM) or female (DSXF) peptides. In Drosophila, an additional SR splicing enhancer component, RBP1, binds to target sites in the splice acceptor preceding the female-specific exon and is essential for efficient splicing of female dsx pre-mRNA . The downstream targets of insect dsx are not well elucidated, however 58 optimal binding sites and associated nearest genes have been identified for D. melanogaster Dmdsx . The red flour beetle Tribolium castaneum Tcdsx has been implicated in oocyte development including Vitellogenins and their associated receptors , while Lepidopteran dsx has been shown to influence expression of pheromone-binding proteins and hexamerin storage proteins .
Orthologs of the dsx gene have currently been identified in seven orders of insects ranging from the primitive Pediculus humanus (human body louse) to several genera of Hymenoptera, however a functional transformer homolog has not always been recovered in these genomes (see  for summary) leading to speculation that some lineages have recruited alternate or additional upstream regulators for dsx . For example, TRA/TRA-2 mediated splicing of dsx has been shown in the Brachyceran flies Ceratitis capitata , Musca domestica  and Lucilia cuprina  yet transformer appears lost in the Nematoceran flies including mosquitoes .
Despite varying primary signals and upstream regulatory mechanisms, male and female-specific DSX peptides of various Diptera including Anastrepha , Ceratitis  and Musca  effected partial masculinization and feminization of genetically female and male D. melanogaster, respectively, when expressed ectopically. This evolutionary conservation is due in part to the retention of two functional protein domains essential for peptide oligomerization: an atypical zinc-finger DNA-binding domain found in multiple members of the DMRT superfamily (DBD/OD1) and an oligomerization domain (OD2) unique to dsx . The DBD/OD1 domain functions to form a dimeric DNA-binding unit that maintains 92 % sequence similarity between Dipteran (D. melanogaster) and Lepidopteran (Bombyx mori) taxa while completely conserving the critical cysteine and histidine residues . The OD2 domain is likely responsible for sex-specific splicing activation or repression of downstream factors , and is modified by sex-specific splicing to maintain both common and male/female-specific portions; the common portion exhibits a greater degree of conservation within and among insect taxa than the C-terminal sex-specific portion [18, 26].
The current Aeadsx gene model  (Fig. 1) spans 450 kb of genomic DNA of supercontig 1.370 and is composed of eight known exons, although nine are likely. Unlike other sequenced Dipteran dsx genes, Aeadsx was found to produce two female-specific isoforms by exon skipping, encoding peptides with alternative C-termini via inclusion of both exons 5a and 5b, or 5b alone. Additionally, analysis of cis-acting elements in Aeadsx revealed a cluster of TRA-2-ISS and RBP1 elements upstream of exon 5a, and Dipteran dsxRE binding sites and PRE elements present only in exon 5b (Fig. 1). Several instances of a motif strongly resembling a potential dsxRE element previously only recovered in the Hymenoptera (NvdsxRE, ) were found within exon and intron 5a. Unlike An. gambiae (and similar to Drosophila) Aeadsx possesses a weak splice acceptor upstream of exon 5b that is activated to splice both female isoforms. Salvemini et al.  hypothesize that regulatory mechanisms governing the sex-specific splicing of the gene in Ae. aegypti are different than in other Diptera including An. gambiae, and that the two female-specific exons were each under the control of a different splicing regulator: A female-specific TRA-like protein acts in females as a splicing activator of exon 5b via dsxRE and PRE elements, while a splice repressor acts on 5a (included by default splicing) in some transcripts. In the males, a male-specific factor may act to repress inclusion of exon 5a via TRA-2-ISS and NvdsxRE elements, while exon 5b is excluded due to lack of female-specific TRA.
Cho et al.  proposed that default female-specific dsx splicing by selective repression of the male isoform (i.e. by the feminizer gene in A. mellifera  and the recently discovered piRNA precursor Fem in B. mori ) is ancestral to holometabolous insects based on its conservation in taxa as phylogenetically distant as A. mellifera and B. mori, and that Diptera possess a derived splicing system where the male form is default and the female form must be ‘splice-activated’ by a TRA/TRA2-like factor. While this appears to be the case in Anastrepha, Drosophila, and An. gambiae doublesex, the data from Salvemini et al.  strongly suggest that the female spliceforms are default in Ae. aegypti; the “strong” exon 5a does not require TRA/TRA2 enhancement, and must be repressed by a male factor. Culicine mosquitoes (inclusive of the genera Aedes and Culex) determine sex at an autosomal locus , while Anopheline mosquitoes possess heteromorphic (XY) sex chromosomes . The latter authors propose that this locus (the M-locus) may either act on intermediary factors or on the dsx gene itself (transformer appears to be either lost or extremely diverged in the mosquitoes , however transformer2 is present) to suppress female-specific dsx splicing and generate the male form. Further, Salvemini et al.  posit that retention of the Hymenopteran-like NvdsxRE elements coupled with Apis-like splicing regulation (and a likely female-specific default splicing) could represent a stably maintained ancestral state in Ae. aegypti exclusive of the rest of known Dipteran doublesex. Recently, analysis of the red flour beetle Tribolium castaneum  revealed three female-specific and one male-specific dsx isoform, with male default splicing occurring via suppression of maternally transferred zygotic TRA protein (required to activate female-specific splicing) by a dominant male factor. This variation in the top-level regulation of dsx among Hymenoptera, Diptera, Lepidoptera, and Coleoptera via upstream factors is in agreement with the theory of Wilkins  stating that the cascade has evolved in reverse order, with the final double-switch gene (doublesex) remaining relatively conserved as additional elements are added and/or neofunctionalization occurs at the upper regulatory levels. As sex determination is critical to insect reproduction, deleterious mutations in dsx could therefore have strong effects on fitness and be selected against. Previous studies have shown the female-specific exon to be evolutionarily conserved [36–38], yet disagree on evolutionary rate comparisons of the common and male-specific portions of the transcript over longer evolutionary time frames. Hughes  found a much greater rate of non-synonymous substitutions within the male-specific region as compared to the common region, while Sobrinho Jr. and de Brito  found nearly equivalent levels of positive selection between the two.
As the production of genetic sexing mosquito strains and molecular methods that create male bias and/or elimination of the female sex are ideal strategies for sterile insect technique , it follows that a conserved sex regulator like doublesex (and transformer) would be optimal molecular targets for such control programs . Elucidating the variable mechanisms by which dsx determines sexual fate in sequenced mosquito lineages is mandatory if progress is to be made towards a control strategy for the world’s deadliest animals. Here we provide full-length gene sequence, sex-specific splicing analyses, and regulatory analysis of the doublesex gene from the southern house mosquito Culex quinquefasciatus (herein Cxqdsx) via RT-PCR and Illumina transcriptome data. Additionally, to discern the strength and location of early evolutionary drivers on doublesex within the Culex pipiens complex, we conduct an evolutionary analysis using Cxqdsx and a newly constructed dsx transcript from Culex pipiens form pipiens (Cxpipdsx). These results provide a comparative platform with which to study sex determination in those mosquitoes with currently sequenced genomes (An. gambiae , Ae. aegypti  and Cx. quinquefasciatus ).
Culex quinquefasciatus mosquitoes were obtained from a colony initiated in 2008 with egg rafts collected from Oahu, Hawaii, USA. Male and female total RNA was extracted separately from twenty adult mosquitoes of each sex using the Qiagen RNeasy Plus Universal Kit (Qiagen, Valencia CA) per manufacturer’s protocol. Prior to extraction, samples were placed in a 2 ml eppendorf tube containing a sterile steel bead + 800 μl Qiazol solution and homogenized for 1 min @ 20Hz on a Qiagen TissueLyser. Contaminant DNA was removed with the TURBO DNA-free DNA Removal Kit (Invitrogen, Carlsbad CA) and first-strand cDNA was generated using the Superscript First-Strand Synthesis System (Invitrogen) per manufacturer’s protocol and diluted to 50 μl in H2O. Four microliters of the cDNA was used in each 25 μl PCR reaction containing 12 μl H2O, 2.5 μl Qiagen Q-solution, 2.5 μl 10× PCR buffer, 0.5 μl dNTPs, 2.5 units AmpliTaq DNA Polymerase (Invitrogen) and 0.5 μl (200 μM final concentration) of each primer. Thermal cycling conditions were as follows: 1 min @ 95 °C, followed by 30 cycles x (30 s @ 94 °C, 30 s @ 50-54 °C primer-specific annealing, 60 s @ 68 °C [120 s for products > 1 kb]), 5 min @ 68 °C final extension.
To recover the complete 5’ end of the transcript, we performed 5’ RACE PCR using the FirstChoice RLM-RACE Kit (Invitrogen) per manufacturer’s protocol using internal gene-specific primers quinqDSX5RACE-GSP1 and quinqDSX5RACE-GSP2 placed adjacent to the OD1 domain. All RT-PCR and RACE-PCR amplicon products were visualized on a 1.5 % agarose gel in TAE buffer and gel-purified using the QIAquick Gel Extraction Kit (Qiagen) prior to cloning via the TOPO TA Cloning Kit (Invitrogen) and PCR-enrichment using the M13 forward/reverse primer pair per manufacturer’s protocol. PCR products were cleaned with ExoSap (Invitrogen) per manufacturer’s protocol, and cycle sequencing was performed by GENEWIZ (South Plainfield, NJ) using the M13 primer pair.
The 3’ end of Cxqdsx was predicted, and the entire gene sequence qualified by mapping the paired-end RNAseq data from NCBI SRA accession SRR991016 generated by Leal et al.  to Cx. quinquefasciatus supercontig 3.59 using the CLC Genomics Workbench (CLC Bio, Aarhus, Denmark) large-gap read mapper (nucleotide similarity score of 95 % over a 95 % read length fraction) and manually examining the output. This process was repeated using the Cx. pipiens f. pipiens paired-end RNAseq library generated by  (see Additional file 2 for details) and the Cx. quinquefasciatus reference generated above to create the full-length gene structure for Cx. pipiens f. pipiens doublesex (Cxpipdsx). To extend the gene model for Aeadsx, we repeated this protocol yet again with the Ae. aegypti NCBI short-read paired-end libraries SRR924024 and SRR789758 and AaegL1.4 supercontig 1.370.
To assess the distribution of the consensus dsxRE (TRA/TRA2) and RBP1 type-b motifs (derived from those of D. melanogaster, An. gambiae and Ae. aegypti), we screened all transcript coding (CDS) sequences corresponding with the Cx. quinquefasciatus Cpip1.3 dataset from VectorBase for their presence. The degenerate motif was broken down into all possible constituents, and each was queried against the CDS dataset with BLASTn (e-val = 999, word_size = 13 [dsxRE] or 7 [RBP1b]). The output was parsed via custom Perl scripts, and transcripts containing six copies of the motif in a 224 bp (for the dsxRE; 546 bp for RBP1b) window were retained. The AhoPro software utility  was used to calculate the probability of observing the motif against a reference dataset of nucleotides randomly generated under a Bernoulli/0-order Markov model.
The synonymous substitutions per synonymous site and nonsynonymous substitutions per nonsynonymous site (Ks and Ka, respectively) and the Ka/Ks ratio were calculated in a pairwise comparison between Cxqdsx and Cxpipdsx using the KaKs Calculator v2.0  under model averaging (MA). We re-calculated these values for each sliding 30 bp window while moving 3 bp (1 amino acid) downstream at a time. To examine base composition of splice acceptor sites, we retrieved 52,278 internal (i.e. exclusive of exon 1) exons with 16 nt of upstream sequence from the CpipJ 1.3 assembly (Vectorbase, ) and calculated the mean number of pyrimidines in the 12 nt preceding the 4 nt splice acceptor.
Results and discussion
Structure and splicing of Cxqdsx
To qualify our Cxqdsx gene model, we mapped the short-read Illumina RNAseq data in NCBI SRA accession SRR991016 generated by Leal et al.  to supercontig 3.59 and manually annotated Cxqdsx. The transcript was well represented in these data, and the structure congrued with our RT-PCR and 5’RACE results in the placement and splicing of all previously described exons including the lack of additional spliceforms in female-specific exon 5 as well as the sequenced 5’ common end of exon 6. Additionally, these data allowed us to define the C-terminus of Cxqdsx, including the full 1,016 bp male-specific/common exon 6 and its splicing over 13,814 bp of intron to a terminal 2,201 bp 7th exon/UTR (Fig. 1, Additional file 3: Figure S1). The final Cxqdsx protein product (Fig. 3) initiates translation in both females and males from the start codon in the common exon 2, and terminates in female mosquitoes at the opal-ochre double stop codons (conserved in Diptera, see ) within exon 5 and in male mosquitoes at a stop codon within exon 6. Exons 6 and 7 are thus transcribed entirely as UTR in the female isoform, as has been shown in other Dipterans including Megaselia scalaris , Anopheles gambiae  and Aedes aegypti .
Completing the Aeadsx gene
To compare the size, structure, intron characteristics and putative promoter regions of our full-length gene model with that of the other sequenced Culicine mosquito, Ae. aegypti, we used publicly available Illumina short-read RNAseq data to discern in-silico the 5’UTR, transcription start site, exon 1 and full 3’UTR of Aeadsx . To predict the 5’ end of Aeadsx, we mapped Illumina short-read RNAseq libraries from NCBI SRA accession SRR789758 to Aedes aegypti strain Liverpool supercontig 1.370 as performed previously and located exon 2 defined by Salvemini et al. . By visual inspection of the mapping, we were able to extend the 2nd exon 472 bp upstream of the start codon, define a splice junction spanning 14,481 bp of intronic sequence, and locate a 1,388 bp 1st exon/5’UTR (Fig. 1, Additional file 5: Figure S3). As RNAseq mapping provides only approximate definition of transcript ends, we searched for a promoter motif within an area +/− 250 bp from the point at which 5’ short-read coverage for exon 1 ceased. We located an initiator element (Inr) of the form YYANWYY at position 109460 of the reverse-complemented supercontig 1.370 and a downstream promoter element (DPE) of the form RGWYV at canonical position +28 from the Inr adenine (Fig. 5), thus providing strong evidence for the Aeadsx transcription start site. As in Cxqdsx, no TATA box was found.
Splice donors and acceptors of the Cxqdsx gene. Coding (exon) sequences are in uppercase text, while the splice donor/acceptor and succeeding/preceeding 12 nucleotides, respectively, are in lowercase. “Exon 4ex” denotes the alternate downstream splice donor of exon 4. Asterisk indicates the splice acceptor site deviates significantly from the genomic mean of 8.58 +/− 1.39 SE (see Methods)
next exon begin
The genera Aedes and Culex are estimated to have diverged ca. 52 Mya . The genome size for Cx. quinquefasciatus currently stands at 540Mbp , while that of Ae. aegypti is estimated to be over twice that size at 1.3Gbp, largely due to the accumulation of transposable elements (TEs) . As TEs are not distributed randomly within chromosomes [52, 53], we assessed the frequency of repetitive elements within the doublesex gene in order to determine whether different classes have invaded the respective dsx genes of Cx. quinquefasciatus and Ae. aegypti. We used CENSOR (http://www.girinst.org/censor/index.php) to scan Cxqdsx introns 2–7 and compared the results to those for Aeadsx intron 2–8  (Additional file 8: Table S2, Additional file 9: Table S3). The two genes contain nearly identical numbers of DNA transposons and similar numbers of LTR retrotransposons, however Aeadsx was found to contain nearly twice as many Non-LTR retrotransposons (or LINEs). These elements persist with great success in eukaryote genomes  and comprise 4 % and 14 % of the transposable elements in the Cx. quinquefasciatus and Ae. aegypti genomes, respectively [43, 44], thus their abundance in doublesex likely reflects the genome-wide pattern.
Regulatory mechanisms of Cxqdsx
All splice junctions of Cxqdsx use conserved GT-AG splice donor/acceptor motifs (Table 1). Interestingly, we find that the number of purines in the polypyrimidine tract of the 3’ splice acceptor preceding the common/male-specific exon 6 (n = 5) deviates significantly from the calculated mean (8.58, +/− 1.39 SE, see Methods) and constitutes a suboptimal splice acceptor. This is contrary to Aeadsx, which is hypothesized to activate a weak splice acceptor upstream of the female-specific exon 5b , and Angdsx which likely relies on activation of the 5’ weak splice donor downstream of exon 5 .
Twenty-two copies of an RBP1 type B motif were present; fourteen copies were located outside of exon 6, however these were represented by eleven different permutations of the consensus sequence. Each 7 nt permutation had a BLASTn e-value of 1.3 when queried against the full Cxqdsx gene sequence, and (in the absence of a clustered distribution) can be expected to occur at least once by chance. Eight copies, however, were clustered in a 546 bp stretch at the 5’ end of the male-specific exon 6. Repeating the protocol used in the TRA/TRA2-like enrichment test above, we find 86 of 19,019 transcripts (0.45 %) contain 8 or more copies of the RBP1b consensus in a 546 bp window. Many of these contigs generated positive results due to tandem repeats however (data summarized in Additional file 12: Table S5). Using AhoPro , we determined the probability of observing this motif in 546 bp of randomly-generated sequence data to be 2.89x10−5. A cluster of Rbp1 binding sites and TRA-2-ISS elements upstream of the “strong” female-specific exon 5a of Aeadsx are hypothesized to manage the differential splicing of this exon while other TRA/TRA2-like elements enhance the “weak” exon 5b  (see Fig. 1). The localization of this RBP1-binding cluster near the “weak” or suboptimal splice acceptor in Cxqdsx exon 6 indicates a SR-like factor may be involved in its splicing. This presents a curious model, as exon 6 is included in both male and female spliceforms. It is thus likely that if exon 6 requires activation by a SR-like factor, it would occur in the male-specific spliceform and facilitate excision of the female-specific exon 5. This would require use of the exon 6 splice acceptor at the expense of exon 5, and could be facilitated by the Rbp1 elements. The functional TRA/TRA2-like factor present in the female would then suffice to maintain incorporation of exon 6 as UTR. Five copies of the TRA-2-ISS motif were found but were not in significant representation. Three copies of the NvTRA element were found, however unlike in Aeadsx that maintains four copies within a cluster in exon 5, two copies were found in intron 4 and one in exon 5. The BLASTn e-value of each 8 bp hit within the search area was 0.37, thus we cannot exclude this result as having occurred simply by chance.
Sequence evolution of Cxqdsx
Our results show that the Cx. quinquefasciatus doublesex gene exhibits sex-specific splicing, as it does in the mosquitoes Ae. aegypti and An. gambie, as well as in other Diptera. Cxqdsx shares characteristics of both Aeadsx (gain of exon 3b, Rbp1 cis-regulatory binding sites) and Angdsx (singular female-specific exon, shared 3’ UTR), as well as a novel spliceform generated from an alternate exon 4 splice donor that appears to occur only in the female. Additionally, we complete the full-length Aeadsx model and identity a putative TATA-less Inr/DPE core promoter region in both Cx. quinquefasciatus and Ae. aegypti mosquito genomes, allowing for future in situ validation and studies of dsx gene transcription.
We find that cis-regulatory splicing regulation of Cxqdsx does not appear to follow either currently described mosquito model, and instead involves activation of a weak splice acceptor of the male-specific/common exon 6, possibly involving a cluster of local Rbp1 binding sites as enhancers. This finding further exemplifies the diversity present in upstream splicing regulation of dsx within mosquitoes, as each of the three genera studied (Anopheles, Aedes and Culex) possess unique regulatory mechanisms despite maintaining TRA/TRA2-like binding sites in the 3’ end of their respective female-specific exons (exon 5b in Aeadsx).
An analysis of peptide evolutionary rates between Cxqdsx and the dsx gene of the closely related Cx. pipiens form pipiens (Cxpipdsx, also generated in this study) shows that the male-specific component of the transcript has evolved at accelerated evolutionary rates relative to the female isoform, and contains sites exhibiting signs of positive selection. This result accentuates the rapid evolution of doublesex within the Culex species complex. Future research defining the degree to which doublesex influences the sexual selection cycle may shed light on the role (if any) that this integral gene plays in incipient speciation within insects.
Availability of supporting data
The nucleotide sequences for the male and female-specific Cxqdsx transcripts have been submitted to GenBank under accession numbers KP033512 and KP033513, respectively. Sequences for male and female-specific Cxpipdsx transcripts have been submitted under accession numbers KP033514 and KP033515.
We are grateful to Linda McCuiston for her unsurpassed expertise in rearing and colonizing the mosquitoes used in our study and to Nicole Wagner at the Rutgers University School of Environmental and Biological Sciences Genome Cooperative for performing our Illumina Sequencing. This work was funded by a New Jersey Mosquito Control Association Daniel M. Jobbins scholarship to DCP and by NE-1043 Multistate funds to DMF.
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