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. 2017 Apr 25;17(2):64. doi: 10.1093/jisesa/iex031

Isolation and Molecular Characterization of the Transformer Gene From Bactrocera cucurbitae (Diptera: Tephritidae)

Ya Luo 1 2,*, Santao Zhao 1 2,*, Jiahui Li 1 2, Peizheng Li 1 2, Rihui Yan 1 2,1 2,3
PMCID: PMC5469387  PMID: 28931159

Abstract

transformer (tra) is a switch gene of sex determination in many insects, particularly in Dipterans. However, the sex determination pathway in Bactrocera cucurbitae (Coquillett), a very destructive pest on earth, remains largely uncharacterized. In this study, we have isolated and characterized one female-specific and two male-specific transcripts of the tra gene (Bcutra) of B. cucurbitae. The genomic structure of Bcutra has been determined and the presence of multiple conserved Transformer (TRA)/TRA-2 binding sites in Bcutra has been found. BcuTRA is highly conservative with its homologues in other tephritid fruit flies. Gene expression analysis of Bcutra at different developmental stages demonstrates that the female transcript of Bcutra appears earlier than the male counterparts, indicating that the maternal TRA is inherited in eggs and might play a role in the regulation of TRA expression. The conservation of protein sequence and sex-specific splicing of Bcutra and its expression patterns during development suggest that Bcutra is probably the master gene of sex determination of B. cucurbitae. Isolation of Bcutra will facilitate the development of a genetic sexing strain for its biological control.

Keywords: Bactrocera cucurbitae (Coquillett), transformer, Bcutra, sex determination, sex-specific splicing

Sex determination that generates female and male dimorphism is a fundamental characteristic of animals and is an essential component of sexual reproduction that is important for the continuity of life. The sex determination of insects has been heavily studied and has been one of the best understood systems (Sanchez 2008, Bachtrog et al. 2014, Bopp et al. 2014). There are several genetic mechanisms that determine the sexual fate among different insects (Saccone et al. 2002, Sanchez 2008, Gempe et al. 2009, Hediger et al. 2010, Shukla and Palli 2012, Verhulst et al. 2013, Kiuchi et al. 2014, Morrow et al. 2014). In general, the primary signals that initiate the sexual fate are perceived by early effectors and thereafter such genetic information are conveyed in early embryonic stages to determine sex and sustained for the sexual identity in the whole life of an organism.

The genetic mechanism of sex determination in Drosophila melanogaster has been extensively examined. In D. melanogaster, the primary signal has been considered to be the ratio of X chromosomes to autosomes (X:A), which actually represents the interaction of the proteins encoded by X-linked elements with the proteins encoded by autosomes (Erickson and Quintero 2007). Sex lethal (Sxl) that acts as a binary switch gene can be switched on or off by the primary signals (Nagoshi et al. 1988). When the X:A ratio is 1:1 (XX:AA), the early female-specific promoter of Sxl is activated, producing an early female-specific SXL protein. This early protein enables the generation of the late SXL protein that can maintain the female-specific splicing of its own pre-mRNA by autoregulation. The late SXL protein also directs the female-specific splicing of its subordinate gene tra, generating a functional TRA protein (Boggs et al. 1987, Nagoshi et al. 1988, Belote et al. 1989, Inoue et al. 1990). TRA and the nonsex-specific Transformer-2 (TRA-2) form a complex to regulate the female-specific splicing of the pre-mRNAs of double sex (dsx) and fruitless (fru), leading to the generation of non-functional FRUF peptide and functional DSXF protein that stimulates the downstream genes to adopt the female pathway of development. On the contrary, when the ratio of X:A is 0.5 (XY:AA, or XO:AA), the early promoter of Sxl is not activated and no early SXL protein is synthesized. Consequently the pre-mRNA of Sxl adopts the male-specific splicing way by default and yields a male-specific transcript with premature in-frame stop codons, resulting in a truncated nonfunctional SXL peptide. The absence of functional SXL protein leads to a cascade of its downstream genes with the male-specific splicing mode, including the generation of nonfunctional TRA and functional male-specific DSXM and FRUM proteins that promote male sexual development (Burtis and Baker 1989, Hoshijima et al. 1991, Tian and Maniatis 1993).

In contrast to D. melanogaster, the primary signal of sex determination in Ceratitis capitata (Wiedemann), a tephritid fruit fly, is an uncharacterized male determining factor (M factor) on Y chromosome (Willhoeft and Franz 1996). It has been proposed that the Y-linked M factor regulates the sex-specific expression of the gene tra by suppressing maternal TRA (Pane et al. 2002). Sxl, however, is expressed in both sexes regardless of the existence of the M factor (Saccone et al. 1998), suggesting that Sxl probably does not have the switch function as in D. melanogaster. In XX females where the M factor is absent, the splicing of the tra homologue of C. capitata (Cctra) is female specific and can produce a functional TRA protein, and CcTRA in turn facilitates its own female-specific splicing through forming an autoregulatory loop (Pane et al. 2002, Gabrieli et al. 2010). The CcTRA/CcTRA-2 complex has been postulated to bind to the putative TRA/TRA-2 binding sites only existed in the male-specific exons and to inhibit the default male-specific splicing mode. Therefore, the female-specific splicing of dsx pre-mRNA is activated, producing a functional female-specific DSXF protein and promoting female sexual development and differentiation. In XY males, however, the presence of the M factor leads to the male-specific splicing of Cctra that generates a truncated non-functional TRA peptide. Without functional TRA, the downstream gene dsx is expressed as functional male-specific DSXM (Pane et al. 2002, Salvemini et al. 2009, Gabrieli et al. 2010).

Although the primary signals of sex determination are different among species, the downstream genes are relatively conserved, such as tra, tra-2, and dsx. tra has also been isolated and characterized from other Dipteran flies, such as Bactrocera dorsalis (Liu et al. 2015, Peng et al. 2015, Laohakieat et al. 2016), B. oleae (Lagos et al. 2007), B. tryoni, B. jarvisi (Morrow et al. 2014), Anastrepha suspensa (Schetelig et al. 2012), A. obliqua (Ruiz et al. 2007), Lucilia cuprina (Concha and Scott 2009), L. sericata, Cochliomyia hominivorax, and C. macellaria (Li et al. 2013). Their molecular organizations and sex-specific splicing patterns are similar to those found in C. capitata. In females, the male-specific exons of those tra genes are spliced out and the resulting transcript is female-specific and can be translated into a functional TRA protein; however, in males, the final transcript includes the male-specific exons with in-frame stop codons and therefore only truncated nonfunctional TRA peptides are translated. The putative TRA/TRA-2 binding sites in the male-specific exons have been found in the tra genes of all the species mentioned above suggest that the regulation of their sex-specific splicing is also similar to the mode in C. capitata, i.e., an autoregulatory mechanism may also occur in those tra genes.

The master role of tra in determining sexual dimorphism makes tra a potential target gene for genetic control of pests. Previous studies have shown that the XX embryos that are supposed to develop into females were reversed to XX pseudomales when the expression of tra was transiently knocked down by microinjecting dsRNA of tra into the embryos of many Dipteran flies, such as C. capitata (Pane et al. 2002), B. dorsalis (Liu et al. 2015, Peng et al. 2015, Laohakieat et al. 2016), B. oleae (Belote et al. 1989), B. tryoni (Raphael et al. 2014), C. macellaria, L. sericata (Li et al. 2013), A. suspensa (Schetelig et al. 2012), and L. cuprina (Concha and Scott 2009). This ability to reverse XX females to pseudomales can be highly advantageous for the application of sterile insect techniques (SIT) in which the male-only release is much more efficient than the release of both sterile males and females (Rendon et al. 2004). Moreover, the region of male-specific exons of tra genes, which acts as an intron in females, has been applied in the control strategy of female-specific lethality in some species and the technology has shown its great potential in genetic control of pests. For example, a female-specific autocidal genetic system has been established in C. capitata through the insertion of a Cctra intron into the gene of a heterologous tetracycline-repressible transactivator (tTAV). In this system, the tTAV transcript is disrupted in males as the Cctra intron is not spliced out while the female tTAV transcript is complete due to the removal of the Cctra intron, and therefore generating a functional tTAV protein that acts as a transactivator and is toxic to cells when it is overexpressed (Fu et al. 2007). Similar systems have established for L. cuprina (Li et al. 2014) and C. hominivorax (Concha et al. 2016).

The melon fly Bactrocera cucurbitae (Diptera: Tephritidae: Dacini) is widely distributed in temperate, subtropical, and tropical regions of the world, causing severe damages in many countries, particularly in China and India. It has been reported that B. cucurbitae damages over 81 host plants and is a very destructive pest of cucurbitaceous vegetables, such as bitter gourd, cucumber, and pumpkin (Dhillon et al. 2005). Many management methods have been applied for its control and one of them is SIT. SIT can be improved by the male-only release that can be achieved by the introduction of female-specific lethality obtained from molecular and genetic modification methods (Rendon et al. 2004, Fu et al. 2007, Li et al. 2014, Concha et al. 2016). However, the mechanism of sex determination and differentiation in B. cucurbitae is still obscure, particularly, the transformer gene of B. cucurbitae has not been identified yet and the role of transformer in sex determination of B. cucurbitae remains unclear.

In this article, we isolate and characterize the transformer gene of B. cucurbitae and examine its expressions at different developmental stages. Phylogenetic tree analysis of BcuTRA demonstrates that it is very similar to the TRA proteins from other tephritid insects. Our results will be beneficial to the understanding of sex determination of B. cucurbitae, the expansion of our knowledge about insect sex determination and the establishment of the theoretical basis for its genetic control in future.

Materials and Methods

Rearing of B. cucurbitae

The melon flies used in this study were collected on City West campus of Hainan University and maintained in the laboratory at 26°C, 70% RH, and a photoperiod of 14:10 (L:D) h. The larvae were reared on artificial diet (Zhou et al. 2016), and the grown larvae were transferred into small plastic boxes with sand before pupation. After 7 d, the pupae were transferred into insect cultivation cages for eclosion. The adults were fed water and a protein-rich food consisting of 1:2 brewer yeast powder/sugar (w/w).

Isolation of Bcutra

To isolate Bcutra, total RNA of B. cucurbitae female adults was prepared using TRI Reagent (Sigma-Aldrich) following the manufacturer’s instructions. The total RNA was treated with DNase I (TaKaRa, Dalian, China), extracted with phenol/chloroform, precipitated with ethanol, and resuspended in nuclease-free water to be used directly for RT-PCR reverse transcription polymerase chain reaction. The first-strand of cDNA was synthesized with an oligo(dT) primer using TaKaRa Prime Script RT-PCR Kit (TaKaRa, Dalian, China). Bcutra cDNA was amplified by PCR with the primers F311+ and R2793- (Supp Table 1 [online only]). These primers were designed on the basis of the alignment of cDNA sequences and protein sequences from B. dorsalis, B. oleae, B. jarvisi, B. tryoni, and B. correcta. This procedure yielded a specific amplification product of 1,165 bp in length. To identify the 5′ and 3′ ends of the Bcutra transcript, 5′ and 3′ RACE rapid amplification of cDNA ends reactions were performed on the 5′ and 3′ RACE cDNA libraries that were made from B. cucurbitae females using SMARTer RACE 5′/3′Kit (Clontech). Two rounds of PCR were performed with the specific primers complimentary to the RACE adaptors and gene-specific primers. For the 3′ RACE, the primer pairs for the first and second (nest) rounds of PCR were F2715+/UPM and F2765+/NUP, respectively. For the 5′ RACE, the primer pairs for the first and second (nest) rounds of PCR were R1625-/UPM and R338-/NUP, respectively.

To determine the male-specific transcripts of Bcutra, similar RT-PCR using male total RNA was carried out with the primers F11+ and R2865-.

To determine the genomic structure of Bcutra, genomic DNA was prepared from adults of B. cucurbitae using Wizard Genomic DNA Purification Kit (Promega), the Bcutra gene was amplified by PCR with the primers F11+ and R2865-.

All PCR products were purified with TaKaRa Mini BEST Agarose Gel DNA Extraction Kit (TaKaRa, Dalian, China) and sequenced by outsourcing (Huada Gene, Shenzhen, China).

Sequence Analysis

Sequence similarity and alignment analysis were performed using BLAST program from NCBI website. Multiple sequence alignment of TRA proteins from different Dipterans was performed using the software DNAMAN (Lynnon Corp.). A neighbor-joining distance tree of TRA proteins was constructed using MEGA6 (Tamura et al. 2013), and the reliability was assessed by the bootstrap method.

Expression of Bcutra Over Development by Semiquantitative RT-PCR

To analyze the temporal expression of Bcutra, total RNAs from different developmental stages (eggs at 0–0.5, 0.5–1, 1–3, 3–6, 6–12, 12–24, and 24–48 h after egg laying; the mixed sexes of the first, second, and third instar larvae, the mixed sexes of pupae; immediately eclosed females and males) were prepared using TRI Reagent (Sigma-Aldrich) and treated with DNase I (TaKaRa, Dalian, China), the synthesis of the first strand of cDNA was performed using TaKaRa Prime Script RT-PCR Kit (TaKaRa, Dalian, China) following the manufacturer’s instructions. Semiquantitative RT-PCRs were performed using the primer pairs F11+/R1573- and F311+/R1063-. The RT-PCR product of the reference gene α-tubulin with the primer pair of α-tubF and α-tubR was used as a control (Liu et al. 2015).

All the primers used in this article are listed in Supp Table 1 [online only].

Results

Gene Structure and Splicing Pattern of Bcutra

To obtain the Bcutra transcription units, we carried out RT-PCR and RACE experiments using total RNA from females and males to obtain full-length cDNAs. Those results revealed that Bcutra is sex-specific splicing (Fig. 1A), similar to tra from other Bactrocera species (Lagos et al. 2007, Morrow et al. 2014, Liu et al. 2015, Laohakieat et al. 2016) and other Dipterans (Verhulst et al. 2010, Geuverink and Beukeboom 2014). B. cucurbitae females contain only one transcript of 1,685 nt (GenBank: KY616908) that comprises a long open reading frame. However, males contain 2 different transcripts (male 1 and male 2) of 2,041 nt (GenBank: KY616909) and 2,424 nt (GenBank: KY616910), both of which are prematurely terminated in that multiple in-frame stop codons exist in the male-specific exons. The full-length Bcutra gene (GenBank: KY616911) is 3,038 bp. The female and male transcripts share the five exons that are designated as exons 1A, 1B, 2, 3, and 4, whereas the exons MS1, MS2, and MS3 are male specific (Fig. 1A).

Fig. 1.

Fig. 1.

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(A) Genomic structure and transcripts of Bcutra. (B) The consensus sequences of the putative TRA/TRA-2 binding sites, TRA-2 ISS and RBP1 binding sites. (A) Exons and introns of Bcutra are represented by boxes and lines, respectively. Exon and intron sizes are represented by the numbers in the boxes and under the lines, respectively. Exons 1A, 1B, 2, 3, and 4 (blue) are common in both females and males. Exons MS1, 2, and 3 are male specific. The start codon and the first stop codon in exons are labeled by black vertical bars. The positions of the putative TRA/TRA-2 binding sites, TRA-2 ISS, and RBP1 binding sites are labeled by purple, yellow, and green vertical bars, respectively. The diagram is not drawn to scale. (B) The conserved sequences are bolded.

The translation start codon is located on the second exon. The TRA protein in females consists of 420 amino acids with a predicted molecular weight of 48.85 kDa and PI value of 11.38. No transmembrane structure was found based on the bioinformatics method TMHMM (Krogh et al. 2001). Both of the male transcripts start translation at the same position with the female transcript but only produce a prematurely truncated and nonfunctional peptide with 66 amino acids as the first stop codon (TAA) exists in MS1 exon at the position of 371 bp (Fig. 1A). Based on the data from similarity analysis of D. melanogaster (Qi et al. 2007), B. dorsalis (Liu et al. 2015, Laohakieat et al. 2016), C. capitata (Pane et al. 2002), Anastrepha spp. (Ruiz et al. 2007), six TRA/TRA-2 binding sites, one TRA-2 intronic splicing silencer (ISS) sequence (Qi et al. 2007) and two type B of RBP1 binding sites (Heinrichs and Baker 1995, Qi et al. 2007) were found in the Bcutra gene (Fig. 1B). Moreover, BcuTRA contains 10 highly conserved amino acids at the region of 284–294, characteristic of the Serine-arginine dipeptides (SR) family that functions in the regulation of protein interactions and specific splicing site recognition (Manley and Tacke 1996, Lagos et al. 2007).

Amino Acid Sequence Alignment and Phylogenetic Analysis of Bcutra

In order to explore the phylogenetic relationship of TRA proteins in Dipterans, including B. cucurbitae, B. dorsalis, B. oleae, B. correcta, B. tryoni, B. jarvisi, A. suspensa, C. capitata, D. melanogaster, multiple sequence alignment and phylogenetic analysis were performed. The results showed that these TRA proteins from Dipteran insects are very similar, exhibiting 71% amino acid identity (Fig. 2A). BcuTRA exhibits the highest degree of similarity with TRA from B. oleae, 74% identical to B. oleae TRA. The secondary similarity occurs between BcuTRA and the TRA protein of B. dorsalis, which is 71% identical. On the other hand, BcuTRA is only 37% identical to the TRA of D. melanogaster. Indeed, Drosophila TRA is considerably shorter than BcuTRA, only 197 amino acids and lack of the amino terminal domain of tephritid TRA.

Fig. 2.

Fig. 2.

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Multiple sequence alignment and phylogenetic tree of TRA proteins. (A) Multiple sequence alignment of TRA proteins in B. cucurbitae, B. dorsalis, B. oleae, B. jarvisi, B. tryoni, B. correcta, A. suspensa, C. capitata, and D. melanogaster. The black-shaded represent the amino acids that are identical in all species, and the gray-shaded represent that the amino acid similarity between species is higher than 75%. The boundary of exons is shown by red vertical lines. SR are within the red box. (B) The neighbor-joining distance tree of Dipteran TRA proteins. The numbers next to the branches represent bootstrap support values from 1,000 replicates. The scale represents the mean character distance. The families are exhibited by color bars on the right. B. cucurbitae (this study) is highlighted with red and with the red triangle. A. obliqua (GenBank: ABW04165.1), A. ludens (GenBank; ABW04176.1), B. dorsalis (GenBank: AME17913.1), B. oleae (GenBank: CAG29241.1), B. tryoni (GenBank: AIK25402.1), B. jarvisi (GenBank: AIK25400.1), C. capitata (GenBank: AAM88673.1), C. macellaria (GenBank: AGE31794.1), C. hominivorax (GenBank: AGE31793.1), D. melanogaster (GenBank: AAB59226.1), D. sechellia (GenBank: AAO38911.1), L. sericata (GenBank: AGE31795.1), L. cuprina (GenBank: ACS34687.2), M. domestica (GenBank: ACY40709.1).

The phylogenetic tree of 15 Dipteran TRA proteins was reconstructed using neighbor-joining method replicated 1,000 times with bootstrap resampling. The results revealed that the phylogenetic relationship for TRA proteins from different Dipteran species is in high agreement with the taxonomic relationship (Fig. 2B). The species of genus Bactrocera, including B. correcta, B. cucurbitae, B. dorsalis, B. jarvisi, B.tryoni, and B. oleae, form one cluster, while Anastrepha species are clustered into the Anastrepha group. The genera of Bactrocera, Anastrepha, and Ceratitis are grouped into the branch of Tephritidae. As expected, TRA from B. cucurbitae and other Bactrocera species are more close to the TRA protein from other tephritid insects than that of Muscidae, Calliphoridae, and Drosophilidae.

Developmental Expression of Bcutra

To determine the expression pattern of Bcutra at various developmental stages of B. cucurbitae, RT-PCR was performed with the primers that amplify a region across the shared first and third exons of Bcutra, generating products of different sizes from the transcripts in females and males (Fig. 3A). As shown in Figure 3B, the female Bcutra transcript appeared from early embryonic stages to pupae as there was always an amplification product of 366 bp detected at these stages. Moreover, the female transcript appeared earlier than the male transcripts as the amplification products of 722 and 1,105 bp from the male transcripts were only detected from the embryos 2 h after egg laying (Fig. 3B). In order to determine the expression of Bcutra in males more precisely, the male-specific transcripts were amplified with the primers of F311+ that is common for the transcripts of males and females and R1063- that is located on the male-specific exon. The results revealed that the male-specific transcripts were able to be detected from the embryos 1 h after egg laying although the amplification bands were a little faint.

Fig. 3.

Fig. 3.

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Expression of Bcutra at different developmental stages. (A) The exon and intron structure of Bcutra. The primers used in (B) and (C) are labeled. (B) The male and female transcripts are amplified using RT-PCR with the primers F11+ and R1573-. (C) The male-specific transcripts are amplified using RT-PCR with the primers F311+ and R1063-. M: DL1000 DNA marker; E0.5-E8: embryos of mixed sexes at 0–0.5, 0.5–1, 1–3, 3–6, 6–12, 12–24, and 24–48 h after egg laying; L1–L3: the first, second and third instar larvae of mixed sexes; P: pupae of mixed sexes; ♂: male adults; ♀: female adults. α-tub: the internal control gene α-tubulin.

Discussion

In this study, the transformer gene of the tephritid pest B. cucurbitae was isolated using RT-PCR and RACE and characterized using multiple methods. Our data show that the transformer gene Bcutra in B. cucurbitae is sex-specific alternative splicing, similar to that of other tephritid fruit flies, such as B. oleae, C. capitata, A.suspensa, Bcutra in females produces a female-specific transcript that encodes a functional TRA protein, whereas Bcutra in males generates two male-specific transcripts that are translated as a nonfunctional truncated peptide in that there are multiple in-frame stop codons in the male transcripts.

The structure of Bcutra and the length of its transcripts in this work are significantly different from the previous transcriptome assembly by Geib and his colleagues in which one female-specific and two male-specific transcripts are predicted (Sim et al. 2015). The first exon predicted by them includes all the sequence of the first exon identified by our work except for the 10 bp (ACATATCTAT) at the very 5′ end. Moreover, the 3′ end of Bcutra in our work is 760 bp less than the one in the previous study. The polyadenylation signal sequence AATAAA exists at the 3′ end of Bcutra isolated from our work and a stretch of poly A is present at the 3′ end of the transcripts from 3′ RACE experiments, suggesting that our results are reliable. Although the female-specific transcripts from two studies are different, the two predicted TRA proteins based on the transcripts have the same sequence of amino acids. In addition, the second exon of the male-specific transcripts are different in size in that the second exon of male 1 and male 2 transcripts in our work are 406 and 946 bp, respectively, their sizes in the previous study, however, are 1,291 and 1,517 bp, respectively.

Our study demonstrates that BcuTRA protein contains a region of Serine-arginine dipeptides (Fig. 2A), suggesting BcuTRA belongs to the SR protein family that is likely to play an important role in protein–protein interactions and splicing control. Although no RNA-binding protein domain exists in BcuTRA, there are TRA/TRA-2 binding sites, TRA-2 ISS sequence, RBP1 binding sites in the male-specific exon region that belongs to the second intron in the female-specific pre-mRNA. The study on TRA-2 of B. cucurbitae has shown that TRA-2 has no sex-specific alternative splicing and belongs to the SR protein family with RNA binding domain (Liu and Wan 2015). These results indicate that BcuTRA may interact with TRA-2 to maintain the splicing of Bcutra, i.e., to autoregulate its own splicing, similar to the splicing of tra in other tephritid fruit flies. In previous studies from B. dorsalis, C. capitata, A. suspensa, et al., TRA has been found to interact with TRA-2 to from TRA/TRA-2 complex to bind the female-specific exon of the downstream gene dsx (Salvemini et al. 2009, Schetelig et al. 2012, Liu et al. 2015). The phylogenetic tree analysis of Dipteran TRA proteins demonstrates that BcuTRA clusters with the TRA proteins from genus Bactrocera and is genetically very close to the genera Ceratitis and Anastrepha. As discussed below, the female-specific Bcutra mRNA appears earlier than the male-specific counterparts (Fig. 3), indicating the existence of maternal Bcutra mRNA in embryos of B. cucurbitae. However, the sex determination of XY embryos is not disturbed by the maternal Bcutra mRNA, suggesting that its function is inhibited. Taken together, we propose the sex determination pathway in B. cucurbitae as shown in Figure 4. In B. cucurbitae, the existence of maternal Bcutra mRNA in XX embryos leads to the translation of functional BcuTRA to initiate the autoregulatory loop of the female-specific splicing of zygotic Bcutra pre-mRNA transcript. The newly translated zygotic BcuTRA and TRA-2 form BcuTRA/TRA-2 complex to maintain the autoregulation of Bcutra and control the female-specific splicing of the downstream gene dsx, which activates female sexual development. In contrast, in XY embryos a Y-linked M factor probably also exists in B. cucurbitae and prevents the expression or disrupts the functions of maternal Bcutra mRNA. Therefore, the initiation of the autoregulatory loop is suppressed and no functional BcuTRA is translated, resulting in the male-specific splicing of zygotic tra and dsx, and consequently male development.

Fig. 4.

Fig. 4.

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Model for sex determination in B. cucurbitae. The functional proteins are represented with color, and the nonfunctional proteins are colorless. Female-specific, male-specific, and common proteins are labeled with blue, green, and orange, respectively.

The expression patterns of Bcutra at different developmental stages demonstrate that the female-specific transcript appears at very early embryonic stages. However, the male-specific transcripts appear at approximately 1 h later after egg laying, which is also later than the female counterpart. This pattern is similar to that of B. dorsalis (Liu et al. 2015). It is actually common as previous studies have shown that the appearance of male-specific transcripts is different in various species. For example, the male-specific transcripts are only detected 4–5 h after egg laying in C. capitata (Pane et al. 2002, Gabrieli et al. 2010) and are detected until the first larval stage in Lucilia cuprina (Concha and Scott 2009). Our research suggests that the upstream regulators of Bcutra may work in the sex determination pathway earlier in B. cucurbitae than in C. capitata and L. cuprina.

The isolation and characterization of Bcutra that reveal some interesting properties of Bcutra have made it become a potential target for genetic control of B. cucurbitae. The conservation of TRA/TRA-2 binding sites, TRA-2 ISS sequence, RBP1 binding sites, and SR region of tephritid tra, including C. capitata and B. cucurbitae, suggests that it is likely to use the strategy of female-specific lethality to genetically control B. cucurbitae. Although RNAi of knocking down Bcutra is not carried out yet, the high conservation of Bcutra indicates that a very similar result of the reversal from XX genotypic females to XX phenotypic males will probably occur when Bcutra is knocked down by RNAi techniques. And therefore, the genetic manipulation of Bcutra can be applied to improve the efficiency of SIT of the pest, or to control the pest by combining other genetic targets, such as the genes that can cause the pest lethal or sterile at a specific developmental stage when they are mutated or their expression is disrupted.

The research about sex determination of tephritid insects has been carried out heavily, however, most studies focus on the downstream regulated genes, such as tra, tra-2 and dsx, the upstream regulators of sex determination have been not very clear. It will be intriguing to study the primary signal of sex determination and its downstream target genes to better understand the whole picture of sex-determining pathway, and therefore providing substantial theoretical basis for the research and applications of sex determination of insects.

Supplementary Material

Supplementary Data
Click here for additional data file. (16.8KB, docx)

Acknowledgments

We thank Shihao Zhou and Yueguan Fu for providing the strain of B. cucurbitae, Yanan Zhang for technical help in rearing B. cucurbitae. This study was supported by Natural Science Fund of Hainan Province (20153069), Scientific Initiation Fund of Hainan University (kyqd1503), Scientific Initiation Fund of Returned Oversea Chinese Scholars from China’s Ministry of Education ([2015]1098), Selected Projects in 2015 on Scientific and Technological Activities Supporting Oversea Chinese Scholars from Ministry of Human Resources and Social Security ([2015]192), and Special Fund for Agro-scientific Research in the Public Interest from Ministry of Agriculture of the People’s Republic of China (201403075).

Supplementary Data

Supplementary data is available at Journal of Insect Science online.

References Cited

  1. Bachtrog D., Mank J. E., Peichel C. L., Kirkpatrick M., Otto S. P., Ashman T. L., Hahn M. W., Kitano J., Mayrose I., Ming R.. et al. 2014. Sex determination: why so many ways of doing it? PLoS Biol. 12: e1001899.. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Belote J. M., McKeown M., Boggs R. T., Ohkawa R., Sosnowski B. A.. 1989. Molecular genetics of transformer, a genetic switch controlling sexual differentiation in Drosophila. Dev. Genet. 10: 143–154. [DOI] [PubMed] [Google Scholar]
  3. Boggs R. T., Gregor P., Idriss S., Belote J. M., McKeown M.. 1987. Regulation of sexual differentiation in D. melanogaster via alternative splicing of RNA from the transformer gene. Cell. 50: 739–747. [DOI] [PubMed] [Google Scholar]
  4. Bopp D., Saccone G., Beye M.. 2014. Sex determination in insects: variations on a common theme. Sexual Dev. 8: 20–28. [DOI] [PubMed] [Google Scholar]
  5. Burtis K. C., Baker B. S.. 1989. Drosophila doublesex gene controls somatic sexual differentiation by producing alternatively spliced mRNAs encoding related sex-specific polypeptides. Cell. 56: 997–1010. [DOI] [PubMed] [Google Scholar]
  6. Concha C., Palavesam A., Guerrero F. D., Sagel A., Li F., Osborne J. A., Hernandez Y., Pardo T., Quintero G., Vasquez M., Keller G. P.. et al. 2016. A transgenic male-only strain of the New World screwworm for an improved control program using the sterile insect technique. BMC Biol. 14: 72.. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Concha C., Scott M. J.. 2009. Sexual development in Lucilia cuprina (Diptera, Calliphoridae) is controlled by the transformer gene. Genetics. 182: 785–798. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dhillon M. K., Singh R., Naresh J. S., Sharma H. C.. 2005. The melon fruit fly, Bactrocera cucurbitae: a review of its biology and management. J. Insect Sci. 5: 40.. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Erickson J. W., Quintero J. J.. 2007. Indirect effects of ploidy suggest X chromosome dose, not the X:A ratio, signals sex in Drosophila. PLoS Biol. 5: e332.. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Fu G., Condon K. C., Epton M. J., Gong P., Jin L., Condon G. C., Morrison N. I., Dafa'alla T. H., Alphey L.. 2007. Female-specific insect lethality engineered using alternative splicing. Nat. Biotechnol. 25: 353–357. [DOI] [PubMed] [Google Scholar]
  11. Gabrieli P., Falaguerra A., Siciliano P., Gomulski L. M., Scolari F., Zacharopoulou A., Franz G., Malacrida A. R., Gasperi G.. 2010. Sex and the single embryo: early deveiopment in the Mediterranean fruit fly, Ceratitis capitata. BMC Dev. Biol. 10: 12.. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gempe T., Hasselmann M., Schiott M., Hause G., Otte M., Beye M.. 2009. Sex determination in honeybees: two separate mechanisms induce and maintain the female pathway. PLoS Biol. 7: e1000222.. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Geuverink E., Beukeboom L. W.. 2014. Phylogenetic distribution and evolutionary dynamics of the sex determination genes doublesex and transformer in insects. Sexual Dev. 8: 38–49. [DOI] [PubMed] [Google Scholar]
  14. Hediger M., Henggeler C., Meier N., Perez R., Saccone G., Bopp D.. 2010. Molecular characterization of the key switch F provides a basis for understanding the rapid divergence of the sex-determining pathway in the housefly. Genetics. 184: 155–170. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Heinrichs V., Baker B. S.. 1995. The Drosophila SR protein RBP1 contributes to the regulation of doublesex alternative splicing by recognizing RBP1 RNA target sequences. EMBO J. 14: 3987–4000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hoshijima K., Inoue K., Higuchi I., Sakamoto H., Shimura Y.. 1991. Control of doublesex alternative splicing by transformer and transformer-2 in Drosophila. Science (New York, N.Y). 252: 833–836. [DOI] [PubMed] [Google Scholar]
  17. Inoue K., Hoshijima K., Sakamoto H., Shimura Y.. 1990. Binding of the Drosophila sex-lethal gene product to the alternative splice site of transformer primary transcript. Nature. 344: 461–463. [DOI] [PubMed] [Google Scholar]
  18. Kiuchi T., Koga H., Kawamoto M., Shoji K., Sakai H., Arai Y., Ishihara G., Kawaoka S., Sugano S., Shimada T., Suzuki Y., Suzuki M. G., Katsuma S.. 2014. A single female-specific piRNA is the primary determiner of sex in the silkworm. Nature. 509: 633–636. [DOI] [PubMed] [Google Scholar]
  19. Krogh A., Larsson B., von Heijne G., Sonnhammer E. L.. 2001. Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J. Mol. Biol. 305: 567–580. [DOI] [PubMed] [Google Scholar]
  20. Lagos D., Koukidou M., Savakis C., Komitopoulou K.. 2007. The transformer gene in Bactrocera oleae: the genetic switch that determines its sex fate. Insect Mol. Biol. 16: 221–230. [DOI] [PubMed] [Google Scholar]
  21. Laohakieat K., Aketarawong N., Isasawin S., Thitamadee S., Thanaphum S.. 2016. The study of the transformer gene from Bactrocera dorsalis and B. correcta with putative core promoter regions. BMC Genetics. 17: 34.. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Li F., Vensko S. P. 2nd, Belikoff E. J., Scott M. J.. 2013. Conservation and sex-specific splicing of the transformer gene in the calliphorids Cochliomyia hominivorax, Cochliomyia macellaria and Lucilia sericata. PloS One. 8: e56303.. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Li F., Wantuch H. A., Linger R. J., Belikoff E. J., Scott M. J.. 2014. Transgenic sexing system for genetic control of the Australian sheep blow fly Lucilia cuprina. Insect Biochem. Mol. Biol. 51: 80–88. [DOI] [PubMed] [Google Scholar]
  24. Liu G., Wan F.. 2015. The gene transformer 2 of Bactrocera fruit flies and its evolvement in insects. J. Environ. Entomol. 37: 742–751. [Google Scholar]
  25. Liu G., Wu Q., Li J., Zhang G., Wan F.. 2015. RNAi-mediated knock-down of transformer and transformer 2 to generate male-only progeny in the oriental fruit fly, Bactrocera dorsalis (Hendel). PloS One. 10: e0128892.. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Manley J. L., Tacke R.. 1996. SR proteins and splicing control. Genes Dev. 10: 1569–1579. [DOI] [PubMed] [Google Scholar]
  27. Morrow J. L., Riegler M., Frommer M., Shearman D. C.. 2014. Expression patterns of sex-determination genes in single male and female embryos of two Bactrocera fruit fly species during early development. Insect Mol. Biol. 23: 754–767. [DOI] [PubMed] [Google Scholar]
  28. Nagoshi R. N., McKeown M., Burtis K. C., Belote J. M., Baker B. S.. 1988. The control of alternative splicing at genes regulating sexual differentiation in D. melanogaster. Cell. 53: 229–236. [DOI] [PubMed] [Google Scholar]
  29. Pane A., Salvemini M., Delli Bovi P., Polito C., Saccone G.. 2002. The transformer gene in Ceratitis capitata provides a genetic basis for selecting and remembering the sexual fate. Development. 129: 3715–3725. [DOI] [PubMed] [Google Scholar]
  30. Peng W., Zheng W., Handler A. M., Zhang H.. 2015. The role of the transformer gene in sex determination and reproduction in the tephritid fruit fly, Bactrocera dorsalis (Hendel). Genetica. 143: 717–727. [DOI] [PubMed] [Google Scholar]
  31. Qi J., Su S., Mattox W.. 2007. The doublesex splicing enhancer components Tra2 and Rbp1 also repress splicing through an intronic silencer. Mol. Cell. Biol. 27: 699–708. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Raphael K. A., Shearman C., Gilchrist A. S., Sved J. A., Morrow J. L., Sherwin W. B., Riegler M., Frommer M.. 2014. Australian endemic pest tephritids: genetic, molecular and microbial tools for improved Sterile Insect Technique. BMC Genetics. 15 Suppl 2: S9.. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Rendon P., McInnis D., Lance D., Stewart J.. 2004. Medfly (Diptera: Tephritidae) genetic sexing: large-scale field comparison of males-only and bisexual sterile fly releases in Guatemala. J. Econ. Entomol. 97: 1547–1553. [DOI] [PubMed] [Google Scholar]
  34. Ruiz M. F., Milano M., Salvemini M., Eirin-Lopez J. M., Perondini A. L., Selivon D., Polito C., Saccone G., Sanchez L.. 2007. The gene transformer of anastrepha fruit flies (Diptera, tephritidae) and its evolution in insects. PloS One. 2: e1239.. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Saccone G., Pane A., Polito L. C.. 2002. Sex determination in flies, fruitflies and butterflies. Genetica. 116: 15–23. [DOI] [PubMed] [Google Scholar]
  36. Saccone G., Peluso I., Artiaco D., Giordano E., Bopp D., Polito L. C.. 1998. The Ceratitis capitata homologue of the Drosophila sex-determining gene sex-lethal is structurally conserved, but not sex-specifically regulated. Development. 125: 1495–1500. [DOI] [PubMed] [Google Scholar]
  37. Salvemini M., Robertson M., Aronson B., Atkinson P., Polito L. C., Saccone G.. 2009. Ceratitis capitata transformer-2 gene is required to establish and maintain the autoregulation of Cctra, the master gene for female sex determination. Int. J. Dev. Biol. 53: 109–120. [DOI] [PubMed] [Google Scholar]
  38. Sanchez L. 2008. Sex-determining mechanisms in insects. Int. J. Dev. Biol. 52: 837–856. [DOI] [PubMed] [Google Scholar]
  39. Schetelig M. F., Milano M., Saccone G., Handler A. M.. 2012. Male only progeny in Anastrepha suspensa by RNAi-induced sex reversion of chromosomal females. Insect Biochem. Mol. Biol. 42: 51–57. [DOI] [PubMed] [Google Scholar]
  40. Shukla J. N., Palli S. R.. 2012. Sex determination in beetles: production of all male progeny by parental RNAi knockdown of transformer. Sci. Rep. 2: 602.. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Sim S. B., Calla B., Hall B., DeRego T., Geib S. M.. 2015. Reconstructing a comprehensive transcriptome assembly of a white-pupal translocated strain of the pest fruit fly Bactrocera cucurbitae. GigaScience. 4: 14.. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Tamura K., Stecher G., Peterson D., Filipski A., Kumar S.. 2013. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol. Biol. Evol. 30: 2725–2729. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Tian M., Maniatis T.. 1993. A splicing enhancer complex controls alternative splicing of doublesex pre-mRNA. Cell. 74: 105–114. [DOI] [PubMed] [Google Scholar]
  44. Verhulst E. C., Lynch A., Bopp D., Beukeboom L. W., van de Zande L.. 2013. A new component of the Nasonia sex determining cascade is maternally silenced and regulates transformer expression. PloS One. 8: e63618.. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Verhulst E. C., van de Zande L., Beukeboom L. W.. 2010. Insect sex determination: it all evolves around transformer. Curr. Opin. Genet. Dev. 20: 376–383. [DOI] [PubMed] [Google Scholar]
  46. Willhoeft U., Franz G.. 1996. Identification of the sex-determining region of the Ceratitis capitata Y chromosome by deletion mapping. Genetics. 144: 737–745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Zhou S., Li L., Fu Y.. 2016. Selection of reference genes in Bactrocera cucurbitae (Coquillett) under high temperature stress. Chin. J. Trop. Crops. 37: 131–135. [Google Scholar]

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