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. 2009 Jul;182(3):785-98.
doi: 10.1534/genetics.109.100982. Epub 2009 May 11.

Sexual development in Lucilia cuprina (Diptera, Calliphoridae) is controlled by the transformer gene

Carolina Concha  1 , Maxwell J Scott
Affiliations

Sexual development in Lucilia cuprina (Diptera, Calliphoridae) is controlled by the transformer gene

Carolina Concha et al. Genetics. 2009 Jul.

Abstract

Insects use an amazing variety of genetic systems to control sexual development. A Y-linked male determining gene (M) controls sex in the Australian sheep blowfly Lucilia cuprina, an important pest insect. In this study, we isolated the L. cuprina transformer (Lctra) and transformer2 (Lctra2) genes, which are potential targets of M. The LCTRA and LCTRA2 proteins are significantly more similar to homologs from tephritid insects than Drosophila. The Lctra transcript is alternatively spliced such that only females make a full-length protein and the presence of six TRA/TRA2 binding sites in the female first intron suggest that Lctra splicing is autoregulated as in tephritids. LCTRA is essential for female development as RNAi knockdown of Lctra mRNA leads to the development of male genitalia in XX adults. Analysis of Lctra expression during development shows that early and midstage male and female embryos express the female form of Lctra and males express only the male form by the first instar larval stage. Our results suggest that an autoregulatory loop sustains female development and that expression of M inhibits Lctra autoregulation, switching its splicing to the male form. The conservation of tra function and regulation in a Calliphorid insect shows that this sex determination system is not confined to Tephritidae. Isolation of these genes is an important step toward the development of a strain of L. cuprina suitable for a genetic control program.

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Figures

F<sc>igure</sc> 1.—
Figure 1.—
Schematic drawing of the genomic organization and the structure of the sex-specific splice variants of Lctra. (A) The top diagram represents the genomic DNA comprising the Lctra locus (to scale). The position of the exons is shown as square boxes, with exons 1, 2, 3, and 4 in red representing common exons to both female and male mRNAs. Exons M1 and M2 in blue represent male specific exons. Introns are represented by solid lines and the 5′- and 3′-untranslated regions are represented by black boxes. Exon and intron sizes are indicated and the translational start and stop sites are marked, but for clarity not all stop sites are shown. The position of putative TRA/TRA2 binding sites within the M2 exon and the first intron is represented by red vertical lines. The splicing patterns of the male and females transcripts are shown below the gene organization diagram (introns are not to scale). (B) Sequence of the six TRA/TRA2 binding sites found in the Lctra genomic DNA sequence and comparison with the D. melanogaster and L. cuprina consensus. (C) Splice donor and acceptor sites for all Lctra introns. The intron 1 “female” donor site is used to make both the female and male 2 transcripts whereas the intron 1 “male” donor site is used only in males. The intron 1 “male” acceptor site is used only to produce the minor male 2 transcript. Both sexes use the intron 1 “common” acceptor site to produce the major transcripts.
F<sc>igure</sc> 2.—
Figure 2.—
Multiple sequence alignment of TRA proteins from L. cuprina, C. capitata, B. oleae, A. obliqua, and D. melanogaster. Identical amino acids are shaded in black while similar amino acids are shaded in gray. Vertical red lines indicate the corresponding locations of the exon/intron boundaries in the L. cuprina and tephritid tra genes. Arrows indicate the conserved motifs that were the basis of the degenerate primers that were used to amplify Lctra cDNA sequence.
F<sc>igure</sc> 3.—
Figure 3.—
Analysis of the expression of Lctra over development by RT–PCR. (A) Position of primers in exons 1 and 4 of Lctra, designed to amplify products of different sizes for female and male transcripts. (B and C) RT–PCR amplification of Lctra on total RNA obtained from different developmental stages of L. cuprina. Male transcripts are 1.5 kb whereas female transcripts are 1.1 kb in size. Stages in B are: E4 early embryos of mixed sexes at 4 hr of development, EF9 midstage female embryos at 9 hr, EM9 midstage male embryos at 9 hr, 1IF female first instar larvae, 1IM male first instar larvae, 3IF female third instar larvae, 3IM male third instar larvae, AF female adults, and AM male adults. In panel C, RNA was isolated from unfertilized eggs (UF) or fertilized precellular embryos at 30–60 min of development (E1) and cDNA prepared either with (+) or without (−) reverse transcriptase.
F<sc>igure</sc> 4.—
Figure 4.—
Injection of Lctra dsRNA into the posterior end of preblastoderm embryos causes female-to-male sex reversal. Phenotypically wild-type females (XX female, A and B) can be recognized from males by a wider interocular distance in the head and by the presence of an ovipositor in the genitalia, whereas males (XY male, A and B) have a pigmented copulatory apparatus with characteristic clasps. Internally, adult females present two ovaries with a large number of eggs (XX female, C) and males present red pigmented testes (XY male, C). Only XX individuals carry the X-linked ZsGreen marker gene. Most frequently, transformed XX males show male genitalia and gonads but conserve the characteristic female head (XX male 1, A, B, C, and D) while a small percentage of the injected XX individuals develop as completely transformed males (XX male 4, A, B, C, and D).
F<sc>igure</sc> 5.—
Figure 5.—
Analysis of the splicing patterns of Lctra and Lcdsx in XX transformed males and wild-type individuals. (A) Control for the RNAi technique; embryos from a cross between Lchsp83-ZsGreen males and wild-type females were injected with dsRNA for ZsGreen. Fluorescence is lost in the posterior end of injected embryos and larvae but not in the anterior end. (B) RT–PCR amplification with Lctra-specific primers on total RNA isolated from heads (h) and bodies (thorax plus abdomen) (b) of transformed XX males presenting male genitalia and female head (XXM1, XXM2, and XXM3), a completely transformed XX male (XXM4) and of a wild-type female and male. The primers used amplify different size products for male (traM) and female transcripts (traF). (C and D) RT–PCR amplification with Lcdsx female and male specific primers, using the same total RNA samples as in B. The sex-specific amplification products are labeled dsxF for female and dsxM for male, respectively. (E) RT–PCR control using α-tubulin-specific primers. The RNAi knockdown of Lctra in XX males sets Lctra splicing in the male mode in the posterior end of injected individuals, which in turn changes the splicing pattern of Lcdsx from the female to the male form.
F<sc>igure</sc> 6.—
Figure 6.—
Multiple sequence alignment of TRA2 proteins in L. cuprina, M. domestica, C. capitata, B. oleae, and D. melanogaster. Identical amino acids are shaded in black while similar amino acids are shaded in gray. Arrows indicate the conserved motifs that were the basis of the degenerate primers that were used to obtain Lctra2 DNA sequence. The RNA recognition motif (RRM) is underlined in red. RNP-1 and RNP-2 are the highly conserved ribonucleoprotein identifier sequences found in RRM motifs. The TRA2 proteins of all these insects are highly conserved in the RRM as well as in the flanking RS domains.
F<sc>igure</sc> 7.—
Figure 7.—
Neighbor-joining tree of insect TRA2 amino acid sequences. The numbers represent bootstrap support values from 1000 replicates. The scale represents the mean character distance.
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