Biology Reference
In-Depth Information
Box 14.1 Release of a Transgenic Strain of a Predatory Mite
The development of a germ-line transformation method in
D. melanogaster
by
Rubin and Spradling
(1982)
instigated this project. Genetic selection of the predatory mite
Metaseiulus occidentalis
had been
successful in improving the field performance of this predator in California almond orchards (
Hoy 1985
).
However, a lack of variability within the species limited the traits that could be modified and transgenic
methods offered the possibility of introducing exogenous genes into the germ line. Because regulatory
issues were perceived to be critical to potential permanent releases of transgenic beneficial arthropods
(
Hoy 1992a,b
, 1995), this project was designed in large part to investigate the regulatory and risk issues
associated with such releases.
In 1990, the availability of useful genes for predatory mites was limited, so a marker gene was constructed
that contained a
lacZ
construct (
E. coli beta-galactosidase
gene) and a
D. melanogaster heatshock 70
promoter
(
Presnail and Hoy 1992
). This construct was developed, in part, because we perceived it to present a low
risk but could still serve as a model to investigate regulatory and risk issues. No TE sequences were included
in the construct out of concerns that TE vectors might be unstable. Efforts to dechorionate and inject
eggs (the method that was successful with
D. melanogaster
) with the plasmid failed, so a novel injection
method was attempted (maternal microinjection). Gravid adult females were injected and 49% of the 405
females survived and deposited eggs; 48 lines were established, with seven of the lines producing larvae that
expressed the
lacZ
construct.
Presnail et al. (1997)
evaluated the stability of four lines by the PCR, Southern
blot analysis, and expression of mRNA. All four colonies were positive by the PCR for the construct, but only
one line was positive by the two Southern blot analyses. The Southern blots indicated that two insertions of
the plasmid had occurred in Line 18. However, all four lines evaluated after 100 generations produced the
appropriate mRNAs, indicating that the
lacZ
construct was being expressed in all four lines.
Transmission of plasmid DNA to eggs by maternal microinjection was found in 70% of all eggs deposited
by the injected females (
Presnail and Hoy 1994
). The persistence and presence of the plasmid DNA in
both injected females and their eggs indicated maternal microinjection could be useful for delivering DNA
into other phytoseiids. However,
Jeyaprakash et al. (1998)
found by Southern-blot analysis that, in 12 new
putatively transgenic lines, at least eight of the lines had extrachromosomally transmitted plasmid DNA.
In five of these eight lines, the plasmid DNA was highly amplified with an estimated 10 million copies
per female. Despite the large number of copies, the extrachromosomal plasmid DNA was lost after 30
generations.
Before release of Line 18, which had two insertions of the construct in the nuclear genome, comparisons
were made between it and wild-type
M. occidentalis
under laboratory conditions to evaluate fitness and
potential risks (
Li and Hoy 1996
). Egg production, hatchability at three temperatures and four relative
humidity conditions, diapause incidence, and proportion of female progeny produced were not different.
Li and Hoy (1996)
also showed that, at high relative humidities and high temperatures, no eggs would
hatch, suggesting that
M. occidentalis
could not survive in Florida's typical summer conditions. (This species
is native to western North America, where summers are dry and most agricultural production occurs
under irrigation.)
Li and Hoy (1996)
also confirmed that the transgenic strain remained an obligatory
predator and would not feed on pollen.
McDermott and Hoy (1997)
evaluated the persistence of
nontransgenic M. occidentalis
in field cages during the summer in Florida and discovered that the predator
populations repeatedly crashed to zero. In addition, analysis with CLIMEX a population growth model
that used climatic factors to determine whether a given poikilothermic species can colonize and persist
in new geographic areas (
http://www.macaulay.ac.uk/dynamo/climex.htm
) also indicated this species
was unlikely to establish in Florida. Furthermore, this predator was released into Florida by the millions
in augmentative biological control efforts directed against spider mites in strawberries in the spring, but
has never established. Therefore, we concluded that release of the transgenic strain would be unlikely to
establish in Florida and therefore would be climatically contained.
