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ARS Home » Southeast Area » Gainesville, Florida » Center for Medical, Agricultural and Veterinary Entomology » Insect Behavior and Biocontrol Research » Research » Publications at this Location » Publication #306863
Title: Male-specific Y-linked transgene markers to enhance biologically-based control of the Mexican fruit fly, Anastrepha ludens (Diptera: Tephritidae)

Author
item MEZA, J.SALVADOR - Chiapas Health Services
item SCHETELIG, MARC - Justus-Liebig University
item ZEPEDA-CISNEROS, C. SILVIA - Chiapas Health Services
item Handler, Alfred - Al

Submitted to: BMC Medical Genetics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/24/2014
Publication Date: 12/1/2014
Citation: Meza, J., Schetelig, M.F., Zepeda-Cisneros, C., Handler, A.M. 2014. Male-specific Y-linked transgene markers to enhance biologically-based control of the Mexican fruit fly, Anastrepha ludens (Diptera: Tephritidae). BMC Medical Genetics. 15(Suppl. 2):1-7.

Interpretive Summary: The creation of transgenic strains of economically important insects for the development of more effective biological control programs is a major goal of our laboratory at CMAVE. A primary goal of transgenic strain development is the introduction of genetic-based marking systems that allow the identification of insects released into the field, and sperm-specific markers that allow the identification of females in the field that have mated with released males. This article describes the development of strains having these transgenic marking systems in the Mexican fruit fly, Anastrepha ludens, which is a highly significant agricultural pest species in Mexico and the southern United States. A transgene construct was created having a green fluorescent protein (EGFP) expressed throughout the body, and a red fluorescent protein (DsRed) expressed only in male sperm due to promoter regulation by the spermatocyte-specific ß2-tubulin promoter. The EGFP marker allows identification of released transgenic males, and the DsRed marker can be detected in the sperm stored in mated females. Both fluorescent protein markers were detected in males having autosomal chromosome transgene integrations, while only the EGFP marker was detected in a male-specific Y-chromosome integration strain (YEGFP). This indicated that the Y-chromosome specifically suppresses expression a male germ-line promoter. The YEGFP strain was then used to create a sexing strain by linking the Y chromosome to the wild type allele of the black pupae (bp+) gene. When introduced into the bp- mutant strain, only males with the dominant-acting bp+ allele have normal brown pupae, while mutant female pupae are black. These pupae can then be sexed using mechanical sorters that distinguish the pupal colors. This has resulted in the development of a mexfly strain that can be sexed during pupation by pupal color, with released sterile males identified in the field by EGFP expression. If the Y-linked ß2-tubulin-DsRed/EGFP transgene can be re-integrated into the linked autosome carrying bp+, sperm-specific Dsred expression is expected, allowing additional use of this essential marker.

Technical 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 ß2-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, Asß2tub-DsRed.T3}, having the polyubiquitin-regulated EGFP body marker, and the ß2-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 Asß2tub-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, Asß2tub-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 the Asß2tub-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.