BMC Genetics最新文献

Background: Females of the Mediterranean fruit fly Ceratitis capitata (Medfly) are major agricultural pests, as they lay eggs into the fruit crops of hundreds of plant species. In Medfly, female sex determination is based on the activation of Cctransformer (Cctra). A maternal contribution of Cctra is required to activate Cctra itself in the XX embryos and to start and epigenetically maintain a Cctra positive feedback loop, by female-specific alternative splicing, leading to female development. In XY embryos, the male determining Maleness-on-the-Y gene (MoY) blocks this activation and Cctra produces male-specific transcripts encoding truncated CcTRA isoforms and male differentiation occurs.

Results: With the aim of inducing frameshift mutations in the first coding exon to disrupt both female-specific and shorter male-specific CcTRA open reading frames (ORF), we injected Cas9 ribonucleoproteins (Cas9 and single guide RNA, sgRNA) in embryos. As this approach leads to mostly monoallelic mutations, masculinization was expected only in G₁ XX individuals carrying biallelic mutations, following crosses of G₀ injected individuals. Surprisingly, these injections into XX-only embryos led to G₀ adults that included not only XX females but also 50% of reverted fertile XX males. The G₀ XX males expressed male-specific Cctra transcripts, suggesting full masculinization. Interestingly, out of six G₀ XX males, four displayed the Cctra wild type sequence. This finding suggests that masculinization by Cas9-sgRNA injections was independent from its mutagenic activity. In line with this observation, embryonic targeting of Cctra in XX embryos by a dead Cas9 (enzymatically inactive, dCas9) also favoured a male-specific splicing of Cctra, in both embryos and adults.

Conclusions: Our data suggest that the establishment of Cctra female-specific autoregulation during the early embryogenesis has been repressed in XX embryos by the transient binding of the Cas9-sgRNA on the first exon of the Cctra gene. This hypothesis is supported by the observation that the shift of Cctra splicing from female to male mode is induced also by dCas9. Collectively, the present findings corroborate the idea that a transient embryonic inactivation of Cctra is sufficient for male sex determination.

Background: The New World Screwworm fly (NWS), Cochliomyia hominivorax, is an ectoparasite of warm-blooded animals and a major pest of livestock in parts of South America and the Caribbean where it remains endemic. In North and Central America it was eradicated using the Sterile Insect Technique (SIT). A control program is managed cooperatively between the governments of the United States and Panama to prevent the northward spread of NWS from infested countries in South America. This is accomplished by maintaining a permanent barrier through the release of millions of sterile male and female flies in the border between Panama and Colombia. Our research team demonstrated the utility of biotechnology-enhanced approaches for SIT by developing a male-only strain of the NWS. The strain carried a single component tetracycline repressible female lethal system where females died at late larval/pupal stages. The control program can be further improved by removing females during embryonic development as larval diet costs are significant.

Results: The strains developed carry a two-component system consisting of the Lucilia sericata bottleneck gene promoter driving expression of the tTA gene and a tTA-regulated Lshid proapoptotic effector gene. Insertion of the sex-specifically spliced intron from the C. hominivorax transformer gene within the Lshid gene ensures that only females die when insects are reared in the absence of tetracycline. In several double homozygous two-component strains and in one "All-in-one" strain that had both components in a single construct, female lethality occurred at the embryonic and/or first instar larval stages when raised on diet without tetracycline. Laboratory evaluation for phenotypes that are relevant for mass rearing in a production facility revealed that most strains had fitness characteristics similar to the wild type J06 strain that is currently reared for release in the permanent barrier. Testing of an "All in one" strain under mass rearing conditions showed that the strain maintained the fitness characteristics observed in small-scale rearing.

Conclusions: The early female lethal strains described here could be selected by the NWS Control Program for testing at large scale in the production facility to enhance the efficiency of the NWS eradication program.

Background: The hopper hAT-family transposable element isolated from the Oriental fruit fly, Bactrocera dorsalis, is distantly related to both the Drosophila hobo element and the Activator element from maize. The original 3120 bp hopper^Bd-Kah element isolated from the Kahuku wild-type strain was highly degenerate and appeared to have a mutated transposase and terminal sequences, while a second 3131 bp element, hopper^Bd-we, isolated from a white eye mutant strain had an intact transposase reading frame and terminal sequences consistent with function.

Results: The hopper^Bd-we element was tested for function by its ability to mediate germline transformation in two dipteran species other than B. dorsalis. This was achieved by creating a binary vector/helper transformation system by linking the hopper^Bd-we transposase reading frame to a D. melanogaster hsp70 promoter for a heat-inducible transposase helper plasmid, and creating vectors marked with the D. melanogaster mini-white⁺ or polyubiquitin-regulated DsRed fluorescent protein markers.

Conclusions: Both vectors were successfully used to transform D. melanogaster, and the DsRed vector was also used to transform the Caribbean fruit fly, Anastrepha suspensa, indicating a wide range of hopper function in dipteran species and, potentially, non-dipteran species. This vector provides a new tool for insect genetic modification for both functional genomic analysis and the control of insect populations.

Background: Anastrepha fraterculus is recognized as a quarantine pest in several American countries. This fruit fly species is native to the American continent and distributed throughout tropical and subtropical regions. It has been reported as a complex of cryptic species, and at least eight morphotypes have been described. Only one entity of this complex, formerly named Anastrepha fraterculus sp. 1, is present in Argentina. Previous cytogenetic studies on this morphotype described the presence of sex chromosome variation identified by chromosomal size and staining patterns. In this work, we expanded the cytological study of this morphotype by analyzing laboratory strains and wild populations to provide information about the frequency and geographic distribution of these sex chromosome variants. We analyzed the mitotic metaphases of individuals from four laboratory strains and five wild populations from the main fruit-producing areas of Argentina, including the northwest (Tucumán and La Rioja), northeast (Entre Ríos and Misiones), and center (Buenos Aires) of the country.

Results: In wild samples, we observed a high frequency of X₁X₁ (0.94) and X₁Y₅ (0.93) karyomorphs, whereas X₁X₂ and X₁Y₆ were exclusively found at a low frequency in Buenos Aires (0.07 and 0.13, respectively), Entre Ríos (0.16 and 0.14, respectively) and Tucumán (0.03 and 0.04, respectively). X₂X₂ and X₂Y₅ karyomorphs were not found in wild populations but were detected at a low frequency in laboratory strains. In fact, karyomorph frequencies differed between wild populations and laboratory strains. No significant differences among A. fraterculus wild populations were evidenced in either karyotypic or chromosomal frequencies. However, a significant correlation was observed between Y₅ chromosomal frequency and latitude.

Conclusions: We discuss the importance of cytogenetics to understand the possible route of invasion and dispersion of this pest in Argentina and the evolutionary forces acting under laboratory conditions, possibly driving changes in the chromosomal frequencies. Our findings provide deep and integral genetic knowledge of this species, which has become of relevance to the characterization and selection of valuable A. fraterculus sp. 1 strains for mass rearing production and SIT implementation.