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reared in the USDA-APHIS quarantine facility for 20 generations without evidence of instability. The stability
is a bit surprising because endogenous putatively intact piggyBac -like elements were found in the PBW ( Wang
et al. 2010 ). During laboratory rearing, no differences in length of time spent in larval instars or the pupal
stage was found, although the transgenic females produced 20% fewer eggs and their hatch rate was 26%
lower ( Miller et al. 2001 ). Based on the Environmental Assessment provided by USDA-APHIS, these moths
were to be contained at the release site in Arizona by sterilization by radiation, having their wings clipped,
and by being placed into a double set of cages isolated from other sources of cotton. Pheromone traps were
to be deployed to capture any transgenic males that escaped, and any cotton was to be destroyed after the
experiment. A release of a high ratio of sterilized moths in the area took place at the end of the experiment,
as well. The goal was to compare the fitness of the transgenic and nontransgenic moths. No journal
publications appear to be available on the results of these contained field trials of a sterilized transgenic
PBW.A transgenic strain containing a red fluorescent protein gene was developed and field releases of
live moths were made in 2006 ( Simmons et al. 2011 ). The injected construct contained the marker gene
regulated by a promoter fragment from a baculovirus nuclear polyhedrosis virus in the piggyBac vector. Four
transgenic lines were produced, but only one appeared to be it. It was reared in a quarantine facility by
USDA-APHIS. The USDA-APHIS issued a release permit after conducting an Environmental Assessment and
notices of the Environmental Assessment were placed in the Federal Register ( Federal Register 2006 ).
The first test was conducted in field cages under USDA permit number 06-150-01r in 2006 in Arizona,
which compared the transgenic strain and the APHIS strain of PBW. Both moth strains were treated with
fluorescent powders, and both were irradiated. The two moth strains were compared for size, response to
pheromone traps, and longevity. No significant differences were found, except the transgenic strain had a
smaller pupal size. In 2007, larger-scale releases into three cotton fields were conducted in Arizona (USDA
permit number 07-015-102r). Approximately 1.1 million sterile moths containing the fluorescent marker
and 1.1 million nontransgenic sterile moths were released into three fields of conventional cotton (non- Bt
cotton). Traps containing the sex pheromone were placed within and outside the fields to determine if the
released transgenic and nontransgenic males dispersed equally and responded to traps in a similar manner.
Mating performance was evaluated by comparing matings between sentinel females tethered at mating
stations with transgenic or nontransgenic males and no significant differences were found in mating
propensity of the two types of males. The traps captured 20% more transgenic moths than nontransgenic
moths, but there was no evidence of a significant difference in “residence” time in the field between the two
strains, and the mean dispersal was greater for the transgenic strain than the nontransgenic strain.
Thus, the transgenic line appeared to be sufficiently it that it could be deployed in an eradication
program. In 2008, > 15 million transgenic PBWs were released (USDA permit 08-105-102rm). Only
transgenic sterile moths were released by air over 2500 acres of cotton in Arizona at a rate of 1 to 2 million
moths per week. The goal was to evaluate the transgenic strain using an eradication program protocol.
Moths in the traps were evaluated for the presence of the transgene by microscopy and DNA was isolated
and evaluated by the PCR to detect the transgene. The transgenic moths were also treated with the red
dye so that comparisons could be made of the efficacy of the marker gene and the dye. The transgene was
reliable under field conditions ( Walters et al. 2012 ).
Box 14.3 Genetic Modification of Aedes aegypti (and Other Mosquitoes) by Insertion
of a Novel Wolbachia Strain (Transinfection)
The mosquito Aedes aegypti is a vector, along with the Asian tiger mosquito Ae. albopictus , of the virus
that causes dengue. No drugs or vaccines are available to manage human cases of dengue, so control
of mosquitoes is the primary disease-management tactic available in the tropics and subtropics around
 
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