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, 7 (10), e1000222

Sex Determination in Honeybees: Two Separate Mechanisms Induce and Maintain the Female Pathway

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Sex Determination in Honeybees: Two Separate Mechanisms Induce and Maintain the Female Pathway

Tanja Gempe et al. PLoS Biol.

Abstract

Organisms have evolved a bewildering diversity of mechanisms to generate the two sexes. The honeybee (Apis mellifera) employs an interesting system in which sex is determined by heterozygosity at a single locus (the Sex Determination Locus) harbouring the complementary sex determiner (csd) gene. Bees heterozygous at Sex Determination Locus are females, whereas bees homozygous or hemizygous are males. Little is known, however, about the regulation that links sex determination to sexual differentiation. To investigate the control of sexual development in honeybees, we analyzed the functions and the regulatory interactions of genes involved in the sex determination pathway. We show that heterozygous csd is only required to induce the female pathway, while the feminizer (fem) gene maintains this decision throughout development. By RNAi induced knockdown we show that the fem gene is essential for entire female development and that the csd gene exclusively processes the heterozygous state. Fem activity is also required to maintain the female determined pathway throughout development, which we show by mosaic structures in fem-repressed intersexuals. We use expression of Fem protein in males to demonstrate that the female maintenance mechanism is controlled by a positive feedback splicing loop in which Fem proteins mediate their own synthesis by directing female fem mRNA splicing. The csd gene is only necessary to induce this positive feedback loop in early embryogenesis by directing splicing of fem mRNAs. Finally, fem also controls the splicing of Am-doublesex transcripts encoding conserved male- and female-specific transcription factors involved in sexual differentiation. Our findings reveal how the sex determination process is realized in honeybees differing from Drosophila melanogaster.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Genomic organization of mRNA producing genes of the SDL.
(A) Diagram of genes within the SDL, which is always heterozygous in females as deduced by high resolution genetic mapping ,. Genes are orientated 5′ to 3′ according to the direction of arrows; the names of functionally characterized genes are underlined. GB30480 corresponds to gene Ex4.8–5.8 . (B) Exon and intron structure diagram of genes encoded at SDL. Exons are shown as boxes and introns by connecting lines. The deduced open reading frames are marked in grey and the presumed start and stop codons are indicated.
Figure 2
Figure 2. Reproductive organ development of 5th instar male and female larvae in the repression analysis of SDL genes.
(A–C) Reproductive organ development of untreated individuals: (A) A pair of normally developed ovaries (ov) and oviducts (od) from an untreated female. (B) A pair of normally differentiated testes from untreated haploid males consisting of densely packed layers of folded testioles. The paired spermducts are not shown. (C) A pair of normally differentiated testes from untreated diploid males consisting of less densely packed layers of folded testioles. The paired spermducts are not shown. (D–G) Repression analysis of gene GB11211 and GB13727. Normally developed gonads of females and haploid males injected with dsRNA devoted to repress the function of gene GB11211 (D–E) and GB13727 (F–G). (H–I) Repression analysis of the fem gene. (H) Pair of underdeveloped testes from a female treated with fem siRNA. The testes of this female individual are covered with oversized epithelial sheaths. The testioles are reduced in length and number when compared with the haploid (B) or diploid (C) males or the pseudomales after csd siRNA injection (J). The shape and course of spermducts appear normal. (I) Normally developed testes from a haploid male injected with fem siRNAs. (J–K) Repression analysis of the csd gene. (J) Pair of fully developed testes from a female treated with csd siRNAs. The number, length, and arrangement of testioles resemble entirely of those dissected from diploid males (C). (K) Normally developed testes from a haploid male injected with csd siRNA. (L–M) Repression analysis of the GB30480 gene. Normally developed gonads of females (L) and haploid males (M) injected with GB30480 dsRNA. Gonads were stained with aceto-orcein (reddish colouring of gonads), which facilitated the dissection process. Scale bars, 1 mm.
Figure 3
Figure 3. Soma and germ line development of female and diploid male late pupae in the knockdown analysis of the fem and the csd gene.
(A–F) Development of the reproductive organ. (A) Normal pair of ovaries, oviducts (od), and unpaired vagina (va) of an untreated female (worker bee). The ovaries are composed of less than five ovarioles (ovl). (B) Normally developed pairs of testes, spermducts (sd), mucus glands (mg), and unpaired endophallus (ep) of a diploid male. The testes consist of hundreds of thickly packed and folded testioles (tl). (C) Male reproductive organ from a female injected with fem siRNAs. The testes are reduced in size and composed of fewer testioles of reduced length. (E) Male reproductive organ from a female treated with csd siRNAs. The testes from these pseudomales are of normal size and structure and appear equivalent to the testes from diploid males (B). (D and F) Normally differentiated reproductive organ from a male treated with fem or csd siRNAs, respectively. Reproductive apparatus was stained with aceto-orcein (reddish colouring), which facilitated the dissection process. Scale bars, 1 mm. (G–L) Differentiation of germ cells in microscopic sections through ovarioles and testicular tubules. (G) Undifferentiated cells in an ovariole of an untreated female. The ovariole is surrounded by an epithelial sheath (white arrowhead). (H) Bundles of spermatids in a testicular tubule (testioles) of a non-injected diploid male. The testicular tubules are composed of spermatocystes containing the spermatids (black arrow) and nurse cells (black arrowhead). (I) Spermatids formed in a fully male differentiated testis of a fem siRNA injected female. (K) Spermatids in a fully male-like developed testis from a csd siRNA treated female. (J and L) Normal testis and germ cell differentiation of fem (J) and csd (L) siRNA injected diploid males. Sections were stained with toluidine blue. Scale bars, 10 µm. (M–S) Development of the inner tibia and tarsus surface of the left hind leg. (M) Normally differentiated pollen brush, pollen comb, and lobe of an untreated female worker. The first tarsal segment displays symmetrically arranged rows of bristles, which are used to brush pollen from the body surface (pollen brush, pb). The upper posterior part of the first tarsal segment forms a lobe (black arrow). Spines at the distal part of the tibia form the pollen comb (grey arrow) in which pollen is detached from the pollen brush. (N) Normally developed tibia and first tarsal segment of non-injected diploid males that lack the symmetrical organization of bristles (pollen brush), the lobe, and the pollen comb. (O) Male differentiated hind leg from a female injected with fem siRNAs. (P) Development of a mosaic intersex upon fem siRNA injections. The posterior part of the first tarsus segment is male, lacks the female-specific lobe (black arrow), and displays bristles in a non-arranged pattern. The anterior part of the tarsal segment is female and shows the symmetrical arrays of bristles. The distal part of the tibia harbours the spines of the pollen comb (grey arrow) indicating a fully developed female structure. (R) Male developed hind leg from a female treated with csd siRNAs. (Q and S) Normally developed hind legs from fem (Q) and csd (S) siRNA injected diploid males. Scale bars, 1 mm.
Figure 4
Figure 4. The processing of fem and Am-dsx transcripts in the response to the knockdowns of the csd and the fem gene induced by RNAi.
(A) The male and female fem and Am-dsx mRNAs of eight 5th larval instar pseudomales that have been injected with csd siRNAs. Fragments corresponding to the fem female (∼350 bp) and male (∼1.6 kb) mRNAs and the Am-dsx female (∼1.4 kb) and male (∼500 bp) mRNAs were amplified by RT-PCR and resolved by agarose gel electrophoresis. The fragments obtained from untreated females and males are shown in the 1st and 2nd lane, respectively. (B) Same analysis as in (A) except that eight pseudomales have been treated with fem instead of csd siRNAs.
Figure 5
Figure 5. Developmental profile of fem mRNA expression.
Fragments corresponding to female (A) and male (B) fem mRNAs were independently amplified by RT-PCR and resolved by agarose gel electrophoresis. The weak ∼1,600 bp fragments observed in reactions devoted to amplify the female-specific fragment correspond to the male mRNAs. Differences in the amount of cDNAs in the different samples were adjusted prior to PCR amplifications. For the embryonic stages the hours after egg deposition are indicated. The early blastoderm is formed ∼12 h after egg deposition. L1 and L5 are 1st and 5th instar larvae, P2 are pupae at medium stage.
Figure 6
Figure 6. The processing of endogenous fem transcripts in response to the injection of Fem encoding mRNA in haploid males.
(A) Fragments corresponding to the female fem mRNAs of individual 72-h-old embryos were amplified by RT-PCR and resolved by agarose gel electrophoresis. The identity of the female fragments was confirmed by nucleotide sequence analysis. The last lane shows the reactions in which the (pl) fem csd-UTR mRNA encoding plasmid (pfemcsd-UTR) was used as a template. The absence of a fragment in this high copy DNA control strongly suggests that our primer oligonucleotides will not amplify fragments corresponding to the injected Fem encoding mRNAs (fem csd-UTR mRNA). (B) Amplified fragments corresponding to the male fem mRNAs on the same set of samples as described in (A).
Figure 7
Figure 7. The regulative hierarchy of honeybee sex determination in relation to other insect model species.
(A) Model for the honeybee sex determination pathway that controls both soma and germ cells. The heterozygous or homo-/hemizygous state of the csd gene determines whether Csd protein is active. Active Csd proteins, derived from different csd alleles in females, are splicing factors that direct the processing into female fem mRNAs. Female fem mRNAs (femF) are producing active Fem proteins that are required to mediate the splicing of Am-dsx pre-mRNA into the female mRNAs. The Fem protein has an additional positive feedback activity that directs the processing of femF mRNAs. Inactive CSD proteins, when derived from homo- or hemizygous csd alleles, result in a splicing of the fem and dsx transcripts, which is the default male state (femM, Am-dsxM). (B) Model for the sex determination pathway in Ceratitis capitata ,. The presence or absence of an unidentified factor M determines sex. In the absence of M the maternal provided Cc-tra gene product establishes an autoregulative loop in which Cc-Tra protein mediates the production of female Cc-tra mRNA. The Cc-Tra protein directs the splicing of Cc-dsx pre-mRNA into the female mode. The presence of M impairs the positive autoregulative loop of the Cc-tra gene products producing a default splicing pattern of Cc-tra transcripts, the male pre-mRNA. The male Cc-dsx mRNA is produced by default. (C) Simplified view of the somatic sex determination hierarchy in D. melanogaster . The X∶A ratio determines whether Sxl is activated. Sxl protein in females is a splicing factor that directs the splicing of tra pre-mRNA into the female mode, resulting in the production of active Tra protein in females. Tra protein mediates the processing of female dsx mRNAs. In the absence of Sxl protein all these regulatory decisions do not occur and the male dsxM is produced by default. The male and female dsx transcripts encode sex-specific transcription factors that have several target genes and are involved in various aspects of sexual differentiation. (D) The evolutionary relationship of the species used in the comparison with their approximate time scale of divergence.

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