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Australian endemic pest tephritids: genetic, molecular and microbial tools for improved Sterile Insect Technique.

Raphael KA, Shearman DC, Gilchrist AS, Sved JA, Morrow JL, Sherwin WB, Riegler M, Frommer M - BMC Genet. (2014)

Bottom Line: Transcriptomes from various species, tissues and developmental stages, to aid in identification of manipulation targets for improving SIT, have been assembled and are in the pipeline.Broad analyses of the microbiome have revealed a metagenome that is highly variable within and across species and defined by the environment.More specific analyses detected Wolbachia at low prevalence in the tropics but absent in temperate regions, suggesting a possible role for this endosymbiont in future control strategies.

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ABSTRACT
Among Australian endemic tephritid fruit flies, the sibling species Bactrocera tryoni and Bactrocera neohumeralis have been serious horticultural pests since the introduction of horticulture in the nineteenth century. More recently, Bactrocera jarvisi has also been declared a pest in northern Australia. After several decades of genetic research there is now a range of classical and molecular genetic tools that can be used to develop improved Sterile Insect Technique (SIT) strains for control of these pests. Four-way crossing strategies have the potential to overcome the problem of inbreeding in mass-reared strains of B. tryoni. The ability to produce hybrids between B. tryoni and the other two species in the laboratory has proved useful for the development of genetically marked strains. The identification of Y-chromosome markers in B. jarvisi means that male and female embryos can be distinguished in any strain that carries a B. jarvisi Y chromosome. This has enabled the study of homologues of the sex-determination genes during development of B jarvisi and B. tryoni, which is necessary for the generation of genetic-sexing strains. Germ-line transformation has been established and a draft genome sequence for B. tryoni released. Transcriptomes from various species, tissues and developmental stages, to aid in identification of manipulation targets for improving SIT, have been assembled and are in the pipeline. Broad analyses of the microbiome have revealed a metagenome that is highly variable within and across species and defined by the environment. More specific analyses detected Wolbachia at low prevalence in the tropics but absent in temperate regions, suggesting a possible role for this endosymbiont in future control strategies.

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Fluoresence in testes and sperm from B. tryoni transformed with vector 1261. (a) A testis dissected from a 1261 transformed fly. Top, white light; bottom, with dsRED Ultra filter. (b) and (c) Confocal images of sperm from a 1261 transformed male (b) and a non-transformed male (c). Sperm were observed using an inverted Zeiss LSM 5 laser scanning confocal microscope. In each case left is the fluorescence image, the right image is bright field.
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Figure 3: Fluoresence in testes and sperm from B. tryoni transformed with vector 1261. (a) A testis dissected from a 1261 transformed fly. Top, white light; bottom, with dsRED Ultra filter. (b) and (c) Confocal images of sperm from a 1261 transformed male (b) and a non-transformed male (c). Sperm were observed using an inverted Zeiss LSM 5 laser scanning confocal microscope. In each case left is the fluorescence image, the right image is bright field.

Mentions: Germ-line transformation is now routine for many non-Drosophilid species and could be used to introduce heritable genetic markers into SIT strains as well as to develop male-only broods. Genetic transformation of B. tryoni has been established [28] using piggyBac as a vector. Flies were marked with the fluorescent proteins EGFP or DsRed under the control of the Drosophila polyubiquitin promoter [29,30], providing the possibility for integrated fluorescent markers. These same vectors have been modified to carry in addition the genes encoding either turboGFP or DsRedExpress markers linked to a C. capitata β2-tubulin spermatogenesis-specific promoter (the 1260 and 1261 vectors respectively), as well as an incorporated attP for site-specific integration, and gypsy insulator elements [31], and have been used to successfully mark sperm in C. capitata [31]. When microinjected into B. tryoni eggs, only one of the two vectors was successfully incorporated into the germ-line (Table 1). The flies had green fluorescence in the body and red fluorescence in dissected testes and sperm (Figure 3). The results demonstrated that the C. capitata β2-tubulin promoter works successfully in the genus Bactrocera. The fluorescent sperm marking make this a useful strain for sperm transfer studies.


Australian endemic pest tephritids: genetic, molecular and microbial tools for improved Sterile Insect Technique.

Raphael KA, Shearman DC, Gilchrist AS, Sved JA, Morrow JL, Sherwin WB, Riegler M, Frommer M - BMC Genet. (2014)

Fluoresence in testes and sperm from B. tryoni transformed with vector 1261. (a) A testis dissected from a 1261 transformed fly. Top, white light; bottom, with dsRED Ultra filter. (b) and (c) Confocal images of sperm from a 1261 transformed male (b) and a non-transformed male (c). Sperm were observed using an inverted Zeiss LSM 5 laser scanning confocal microscope. In each case left is the fluorescence image, the right image is bright field.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4255846&req=5

Figure 3: Fluoresence in testes and sperm from B. tryoni transformed with vector 1261. (a) A testis dissected from a 1261 transformed fly. Top, white light; bottom, with dsRED Ultra filter. (b) and (c) Confocal images of sperm from a 1261 transformed male (b) and a non-transformed male (c). Sperm were observed using an inverted Zeiss LSM 5 laser scanning confocal microscope. In each case left is the fluorescence image, the right image is bright field.
Mentions: Germ-line transformation is now routine for many non-Drosophilid species and could be used to introduce heritable genetic markers into SIT strains as well as to develop male-only broods. Genetic transformation of B. tryoni has been established [28] using piggyBac as a vector. Flies were marked with the fluorescent proteins EGFP or DsRed under the control of the Drosophila polyubiquitin promoter [29,30], providing the possibility for integrated fluorescent markers. These same vectors have been modified to carry in addition the genes encoding either turboGFP or DsRedExpress markers linked to a C. capitata β2-tubulin spermatogenesis-specific promoter (the 1260 and 1261 vectors respectively), as well as an incorporated attP for site-specific integration, and gypsy insulator elements [31], and have been used to successfully mark sperm in C. capitata [31]. When microinjected into B. tryoni eggs, only one of the two vectors was successfully incorporated into the germ-line (Table 1). The flies had green fluorescence in the body and red fluorescence in dissected testes and sperm (Figure 3). The results demonstrated that the C. capitata β2-tubulin promoter works successfully in the genus Bactrocera. The fluorescent sperm marking make this a useful strain for sperm transfer studies.

Bottom Line: Transcriptomes from various species, tissues and developmental stages, to aid in identification of manipulation targets for improving SIT, have been assembled and are in the pipeline.Broad analyses of the microbiome have revealed a metagenome that is highly variable within and across species and defined by the environment.More specific analyses detected Wolbachia at low prevalence in the tropics but absent in temperate regions, suggesting a possible role for this endosymbiont in future control strategies.

View Article: PubMed Central - HTML - PubMed

ABSTRACT
Among Australian endemic tephritid fruit flies, the sibling species Bactrocera tryoni and Bactrocera neohumeralis have been serious horticultural pests since the introduction of horticulture in the nineteenth century. More recently, Bactrocera jarvisi has also been declared a pest in northern Australia. After several decades of genetic research there is now a range of classical and molecular genetic tools that can be used to develop improved Sterile Insect Technique (SIT) strains for control of these pests. Four-way crossing strategies have the potential to overcome the problem of inbreeding in mass-reared strains of B. tryoni. The ability to produce hybrids between B. tryoni and the other two species in the laboratory has proved useful for the development of genetically marked strains. The identification of Y-chromosome markers in B. jarvisi means that male and female embryos can be distinguished in any strain that carries a B. jarvisi Y chromosome. This has enabled the study of homologues of the sex-determination genes during development of B jarvisi and B. tryoni, which is necessary for the generation of genetic-sexing strains. Germ-line transformation has been established and a draft genome sequence for B. tryoni released. Transcriptomes from various species, tissues and developmental stages, to aid in identification of manipulation targets for improving SIT, have been assembled and are in the pipeline. Broad analyses of the microbiome have revealed a metagenome that is highly variable within and across species and defined by the environment. More specific analyses detected Wolbachia at low prevalence in the tropics but absent in temperate regions, suggesting a possible role for this endosymbiont in future control strategies.

Show MeSH
Related in: MedlinePlus