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. 2019 Aug 27;9(1):12408.
doi: 10.1038/s41598-019-47509-6.

Production of WW males lacking the masculine Z chromosome and mining the Macrobrachium rosenbergii genome for sex-chromosomes

Tom Levy  1 , Ohad Rosen  2 , Rivka Manor  1 , Shahar Dotan  2 , Dudu Azulay  2 , Anna Abramov  2 , Menachem Y Sklarz  3 , Vered Chalifa-Caspi  3 , Kobi Baruch  4 , Assaf Shechter  2 , Amir Sagi  5   6
Affiliations

Production of WW males lacking the masculine Z chromosome and mining the Macrobrachium rosenbergii genome for sex-chromosomes

Tom Levy et al. Sci Rep. .

Abstract

The cultivation of monosex populations is common in animal husbandry. However, preselecting the desired gender remains a major biotechnological and ethical challenge. To achieve an efficient biotechnology for all-female aquaculture in the economically important prawn (Macrobrachium rosenbergii), we achieved - for the first time - WW males using androgenic gland cells transplantation which caused full sex-reversal of WW females to functional males. Crossing the WW males with WW females yielded all-female progeny lacking the Z chromosome. We now have the ability to manipulate - by non-genomic means - all possible genotype combinations (ZZ, WZ and WW) to retain either male or female phenotypes and hence to produce monosex populations of either gender. This calls for a study of the genomic basis underlying this striking sexual plasticity, questioning the content of the W and Z chromosomes. Here, we report on the sequencing of a high-quality genome exhibiting distinguishable paternal and maternal sequences. This assembly covers ~ 87.5% of the genome and yielded a remarkable N50 value of ~ 20 × 106 bp. Genomic sex markers were used to initiate the identification and validation of parts of the W and Z chromosomes for the first time in arthropods.

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Conflict of interest statement

Patents regarding Functional sex-reversal of decapod crustacean female (PCT/IL2015/051096), as well as the production of WW homogametic males (PCT/IL2016/051219) are pending.

Figures

Figure 1
Figure 1
Two-phase biotechnology to produce all-female M. rosenbergii populations. (A) A single injection of AG cell suspension caused full sex-reversal of WZ females into WZ neo-males; the progeny of these WZ neo-males crossed with WZ females included 25% WW females. (B) Injection of AG cell suspension into WW females caused sex-reversal into WW neo-males; the progeny of these WW neo-males crossed with WW females yielded WW all-female populations.
Figure 2
Figure 2
Phenotypic and genotypic characterization of M. rosenbergii WW neo-males. (A) Representative male morphotypes from an adult WW neo-male population: blue-claw (BC), orange-claw (OC) and two small males (SM I and II). Bars = 5 cm. (B) Genomic sex markers of normal female (WZ genotype), normal male (ZZ genotype) and four neo-male individuals of the three male morphotypes depicted in A (all with the WW genotype). A 100 bp DNA ladder is given.
Figure 3
Figure 3
Proof, using sex specific genomic markers, of the existence of all possible genotype-phenotype combinations in M. rosenbergii. The gel showing PCR amplification of sex-specific genomic markers (W – top and Z – bottom) shows, from left to right: WZ female, WW female, ZZ female, WZ male, WW male, and ZZ male. A 100 bp DNA ladder is given.
Figure 4
Figure 4
Flow cytometry for the estimation of genome size. Representative histogram of cell fluorescence relative intensity of M. rosenbergii (A) and H. sapiens (B). The Geo mean of each histogram was recorded and used to calculate the genome size of M. rosenbergii relative to the known reference, H. sapiens.
Figure 5
Figure 5
Search for W- and Z-associated scaffolds as a step towards sequencing of the sex chromosomes. Pathways to identify W- and Z-associated scaffolds (starting from our previous genomic sex markers) are indicated with blue arrows, while those to extend validated W- and Z-associated scaffolds are indicated with green arrows.
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