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Male-specific Y-linked transgene markers to enhance biologically-based control of the Mexican fruit fly, Anastrepha ludens (Diptera: Tephritidae).

Meza JS, Schetelig MF, Zepeda-Cisneros CS, Handler AM - BMC Genet. (2014)

Bottom Line: Autosomal insertion lines effectively expressed both markers, but a single Y-linked insertion (YEGFP strain) expressed only PUbnlsEGFP.The PUbnlsEGFP marker remained effective however, and the YEGFP strain was used to create a sexing strain by translocating the wild type allele of the black pupae (bp+) gene onto the Y, which was then introduced into the bp- mutant strain.A Y-linked insertion of the pBXL{PUbnlsEGFP, Asβ2tub-DsRed.T3} transformation vector in A. ludens resulted in male-specific expression of the EGFP fluorescent protein marker, and was integrated into a black pupae translocation sexing strain (T(YEGFP/bp+), allowing the identification of male adults when used in sterile male release programs for population control.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Background: Reliable marking systems are critical to the prospective field release of transgenic insect strains. This is to unambiguously distinguish released insects from wild insects in the field that are collected in field traps, and tissue-specific markers, such as those that are sperm-specific, have particular uses such as identifying wild females that have mated with released males. For tephritid fruit flies such as the Mexican fruit fly, Anastrepha ludens, polyubiquitin-regulated fluorescent protein body markers allow transgenic fly identification, and fluorescent protein genes regulated by the spermatocyte-specific β2-tubulin promoter effectively mark sperm. For sterile male release programs, both marking systems can be made male-specific by linkage to the Y chromosome.

Results: An A. ludens wild type strain was genetically transformed with a piggyBac vector, pBXL{PUbnlsEGFP, Asβ2tub-DsRed.T3}, having the polyubiquitin-regulated EGFP body marker, and the β2-tubulin-regulated DsRed.T3 sperm-specific marker. Autosomal insertion lines effectively expressed both markers, but a single Y-linked insertion (YEGFP strain) expressed only PUbnlsEGFP. This insertion was remobilized by transposase helper injection, which resulted in three new autosomal insertion lines that expressed both markers. This indicated that the original Y-linked Asβ2tub-DsRed.T3 marker was functional, but specifically suppressed on the Y chromosome. The PUbnlsEGFP marker remained effective however, and the YEGFP strain was used to create a sexing strain by translocating the wild type allele of the black pupae (bp+) gene onto the Y, which was then introduced into the bp- mutant strain. This allows the mechanical separation of mutant female black pupae from male brown pupae, that can be identified as adults by EGFP fluorescence.

Conclusions: A Y-linked insertion of the pBXL{PUbnlsEGFP, Asβ2tub-DsRed.T3} transformation vector in A. ludens resulted in male-specific expression of the EGFP fluorescent protein marker, and was integrated into a black pupae translocation sexing strain (T(YEGFP/bp+), allowing the identification of male adults when used in sterile male release programs for population control. A unique observation was that expression of the Asβ2tub-DsRed.T3 sperm-specific marker, which was functional in autosomal insertions, was specifically suppressed in the Y-linked insertion. This may relate to the Y chromosomal regulation of male-specific germ-line genes in Drosophila.

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Y-linked and autosomal fluorescent marker expression in A. ludens transformed with pBXL{PUbnlsEGFP, Asβ2tub-DsRed.T3}. The brightfield (BF; A, D, G) and epifluorescent EGFP (GFP2; B, E, H), and DsRed (TXR; C, F, I) phenotypes of: a YEGFP male (left) and female (right) shown in panels A, B, and C; an autosomal insertion (unmapped) strain male (left) and female (right) shown in panels D, E, and F; and testes from a YEGFP and autosomal insertion strain male shown in panels G, H, and I. See Methods for details on epifluorescent microscopy and filter sets.
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Figure 1: Y-linked and autosomal fluorescent marker expression in A. ludens transformed with pBXL{PUbnlsEGFP, Asβ2tub-DsRed.T3}. The brightfield (BF; A, D, G) and epifluorescent EGFP (GFP2; B, E, H), and DsRed (TXR; C, F, I) phenotypes of: a YEGFP male (left) and female (right) shown in panels A, B, and C; an autosomal insertion (unmapped) strain male (left) and female (right) shown in panels D, E, and F; and testes from a YEGFP and autosomal insertion strain male shown in panels G, H, and I. See Methods for details on epifluorescent microscopy and filter sets.

Mentions: YEGFP vector remobilization. Segregation analysis of lines transformed with the piggyBac transformation vector, pBXL{PUbnlsEGFP, Asβ2tub-DsRed.T3} (see Additional file 1) [13], indicated that one line, YEGFP, was Y-linked due to PUbnlsEGFP fluorescent marker expression being limited to males, and segregation analysis showing male-specific inheritance (Figure 1). However, the sperm-specific expression of Asβ2tub-DsRed.T3, observed in four other autosomal insertion lines, was not observed in the Y-linked line (Fig. 1A-C). The structural integrity of the Asβ2tub-DsRed.T3 vector construct in YEGFP was verified by PCR sequencing (see Additional file 2), indicating that this was not due to a mutation or rearrangement.


Male-specific Y-linked transgene markers to enhance biologically-based control of the Mexican fruit fly, Anastrepha ludens (Diptera: Tephritidae).

Meza JS, Schetelig MF, Zepeda-Cisneros CS, Handler AM - BMC Genet. (2014)

Y-linked and autosomal fluorescent marker expression in A. ludens transformed with pBXL{PUbnlsEGFP, Asβ2tub-DsRed.T3}. The brightfield (BF; A, D, G) and epifluorescent EGFP (GFP2; B, E, H), and DsRed (TXR; C, F, I) phenotypes of: a YEGFP male (left) and female (right) shown in panels A, B, and C; an autosomal insertion (unmapped) strain male (left) and female (right) shown in panels D, E, and F; and testes from a YEGFP and autosomal insertion strain male shown in panels G, H, and I. See Methods for details on epifluorescent microscopy and filter sets.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4255795&req=5

Figure 1: Y-linked and autosomal fluorescent marker expression in A. ludens transformed with pBXL{PUbnlsEGFP, Asβ2tub-DsRed.T3}. The brightfield (BF; A, D, G) and epifluorescent EGFP (GFP2; B, E, H), and DsRed (TXR; C, F, I) phenotypes of: a YEGFP male (left) and female (right) shown in panels A, B, and C; an autosomal insertion (unmapped) strain male (left) and female (right) shown in panels D, E, and F; and testes from a YEGFP and autosomal insertion strain male shown in panels G, H, and I. See Methods for details on epifluorescent microscopy and filter sets.
Mentions: YEGFP vector remobilization. Segregation analysis of lines transformed with the piggyBac transformation vector, pBXL{PUbnlsEGFP, Asβ2tub-DsRed.T3} (see Additional file 1) [13], indicated that one line, YEGFP, was Y-linked due to PUbnlsEGFP fluorescent marker expression being limited to males, and segregation analysis showing male-specific inheritance (Figure 1). However, the sperm-specific expression of Asβ2tub-DsRed.T3, observed in four other autosomal insertion lines, was not observed in the Y-linked line (Fig. 1A-C). The structural integrity of the Asβ2tub-DsRed.T3 vector construct in YEGFP was verified by PCR sequencing (see Additional file 2), indicating that this was not due to a mutation or rearrangement.

Bottom Line: Autosomal insertion lines effectively expressed both markers, but a single Y-linked insertion (YEGFP strain) expressed only PUbnlsEGFP.The PUbnlsEGFP marker remained effective however, and the YEGFP strain was used to create a sexing strain by translocating the wild type allele of the black pupae (bp+) gene onto the Y, which was then introduced into the bp- mutant strain.A Y-linked insertion of the pBXL{PUbnlsEGFP, Asβ2tub-DsRed.T3} transformation vector in A. ludens resulted in male-specific expression of the EGFP fluorescent protein marker, and was integrated into a black pupae translocation sexing strain (T(YEGFP/bp+), allowing the identification of male adults when used in sterile male release programs for population control.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Background: Reliable marking systems are critical to the prospective field release of transgenic insect strains. This is to unambiguously distinguish released insects from wild insects in the field that are collected in field traps, and tissue-specific markers, such as those that are sperm-specific, have particular uses such as identifying wild females that have mated with released males. For tephritid fruit flies such as the Mexican fruit fly, Anastrepha ludens, polyubiquitin-regulated fluorescent protein body markers allow transgenic fly identification, and fluorescent protein genes regulated by the spermatocyte-specific β2-tubulin promoter effectively mark sperm. For sterile male release programs, both marking systems can be made male-specific by linkage to the Y chromosome.

Results: An A. ludens wild type strain was genetically transformed with a piggyBac vector, pBXL{PUbnlsEGFP, Asβ2tub-DsRed.T3}, having the polyubiquitin-regulated EGFP body marker, and the β2-tubulin-regulated DsRed.T3 sperm-specific marker. Autosomal insertion lines effectively expressed both markers, but a single Y-linked insertion (YEGFP strain) expressed only PUbnlsEGFP. This insertion was remobilized by transposase helper injection, which resulted in three new autosomal insertion lines that expressed both markers. This indicated that the original Y-linked Asβ2tub-DsRed.T3 marker was functional, but specifically suppressed on the Y chromosome. The PUbnlsEGFP marker remained effective however, and the YEGFP strain was used to create a sexing strain by translocating the wild type allele of the black pupae (bp+) gene onto the Y, which was then introduced into the bp- mutant strain. This allows the mechanical separation of mutant female black pupae from male brown pupae, that can be identified as adults by EGFP fluorescence.

Conclusions: A Y-linked insertion of the pBXL{PUbnlsEGFP, Asβ2tub-DsRed.T3} transformation vector in A. ludens resulted in male-specific expression of the EGFP fluorescent protein marker, and was integrated into a black pupae translocation sexing strain (T(YEGFP/bp+), allowing the identification of male adults when used in sterile male release programs for population control. A unique observation was that expression of the Asβ2tub-DsRed.T3 sperm-specific marker, which was functional in autosomal insertions, was specifically suppressed in the Y-linked insertion. This may relate to the Y chromosomal regulation of male-specific germ-line genes in Drosophila.

Show MeSH
Related in: MedlinePlus