Identification and characterization of a Masculinizer homologue in the diamondback moth, Plutella xylostella

Abstract

Recently, a novel sex-determination system was identified in the silkworm (Bombyx mori) in which a piwi-interacting RNA (piRNA) encoded on the female-specific W chromosome silences a Z-linked gene (Masculinizer) that would otherwise initiate male sex-determination and dosage compensation. Masculinizer provides various opportunities for developing improved genetic pest management tools. A pest lepidopteran in which a genetic pest management system has been developed, but which would benefit greatly from such improved designs, is the diamondback moth, Plutella xylostella. However, Masculinizer has not yet been identified in this species. Here, focusing on the previously described 'masculinizing' domain of B. mori Masculinizer, we identify P. xylostella Masculinizer (PxyMasc). We show that PxyMasc is Z-linked, regulates sex-specific alternative splicing of doublesex and is necessary for male survival. Similar results in B. mori suggest this survival effect is possibly through failure to initiate male dosage compensation. The highly conserved function and location of this gene between these two distantly related lepidopterans suggests a deep role for Masculinizer in the sex-determination systems of the Lepidoptera.

Keywords: Plutella xylostella; doublesex; masculinizer; diamondback moth; dosage compensation; gene drive; sex determination.

Figure 1

Characterizing sex‐alternative splicing of doublesex (dsx) in the diamondback moth (DBM). (A) Results of Reverse‐Transcription‐PCR (RT‐PCR) conducted on male and female pupal cDNA using primers specific to the exons flanking the sex‐alternatively spliced region (exons 2 and 5 in B) of dsx. No template control (NTC) shown by (−). Visible bands were cloned and sequenced identifying a single male‐specific transcript (M) and four female transcripts (F1–F4) (B). Intron/exon structure of the dsx gene within this region, revealed by sequencing RT‐PCR products from A, relative to genomic sequence (G). Dotted lines represent alternative splice sites within exons. The male transcript includes the shared exons (2 and 5) but excludes the internal female‐specific exons (3 and 4). Female transcripts all contained both the shared exons and various combinations of the alternatively spliced female‐specific exons. For transcripts, coding region is shown in black and 3′ untranslated region in white. Sequences of each transcript are available in the Supporting Information Figure S1. Panel C shows dsx splicing patterns in individual embryos following injection with double‐stranded RNA (dsRNA) targeting either the Plutella xylostella Masculinizer (PxyMasc) transcript (upper row) or the Anemonia majano Cyan (AmCyan) control (lower row). Second‐from‐right lane in the AmCyan row is the NTC. Panel C consists of two cropped images from a single gel. For reference, the uncropped image can be found in the Supporting Information Figure S4. PCR images shown are of representative samples from each treatment.

Figure 2

Temporal analysis of Plutella xylostella Masculinizer (PxyMasc) and doublesex (dsx) expression/splicing patterns in early eggs. Individual eggs were collected within 5 min of being laid and allowed to develop for 3, 6 and 24 h before being sampled (frozen in liquid nitrogen, RNA extracted, cDNA synthesized). Samples were used as template for paired PxyMasc/dsx PCR. A minimum of 25 eggs were obtained at each time point with the images above displaying representative samples from each group. Panel A shows results of dsx PCR, panel B shows results of PxyMasc PCR (PCRs in the same lanes in these two panels were run using the same sample, ie cDNA from the same egg), panel C shows controls run using sexed male (M) and female (F) pupal cDNA as well as a no template control (−). At 3 h postlaying, all eggs show female dsx splicing and no PxyMasc transcript is observable. At 6 h postlaying all individuals are showing both male (c. 200 bp) and female (c. 600 & 450 bp) dsx bands and all show full PxyMasc expression (c. 1200 bp) as well as a smaller PxyMasc transcript (c. 200 bp, Fig. 3B). At 24 h postlaying, samples showed either male dsx splicing (first three samples) or female dsx splicing (second three samples) with PxyMasc expression only remaining in those individuals showing male dsx bands.

Figure 3

Characterization of Plutella xylostella Masculinizer (PxyMasc) gene structure. PxyMasc gene structure (panel A) was determined through 5′ and 3′ rapid amplification of cDNA ends from exons 5 and 12/13, respectively, followed by RT‐PCR of the intervening region. These analyses revealed the presence of the CCCH‐tandem (Cysteine‐Cysteine‐Cysteine‐Histidine) zinc finger motifs (ZF1 and ZF2) and the masculinizing ‘cysteine‐cysteine domain’ (CAEC) indicative of other Masc homologues (details provided in panel D). RT‐PCR revealed a secondary PxyMasc transcript (panel B) where exons 5–12 were excised (exons 4–13 directly joined). This allowed a small region within exon 13 to be included within the coding sequence. Putative coding regions are shown in black, untranslated regions in white. Analysis revealed that the previously unannotated coding exon 2 contained amino acid sequences homologous to the CCCH‐tandem zinc finger motifs identified in Bombyx mori Masc (alignment shown in panel C). Here, the CCCH conserved amino acids are highlighted in red. Panel D shows alignment of the integral masculinizing cysteine‐cysteine domain (found in exon 5) from lepidopterans where Masc homologues have been identified (sequences other than Plutella adapted from Katsuma et al., 2015). The integral cysteine‐cysteine region is outlined in black. Full transcript sequence is given in Fig. 3.

Figure 4

Genomic copy number of Plutella xylostella Masculinizer (PxyMasc) in male and female diamondback moths (DBM) estimated by quantitative PCR (qPCR) on genomic DNA template. Relative quantification (2^−ΔΔCq) of PxyMasc and a known Z‐linked positive control (kettin) in male (black bars; n = 10) and female (grey bars; n = 10) DBM pupae. Bars represent mean (2^−ΔΔCq) values and error bars represent standard error. A known autosomal gene (defensin) was used as a reference gene during analysis.