Contents - Faperta

296 Biotechnological Approaches for Pest Management and Ecological Sustainability
of M. occidentalis by inserting the needle through the cuticle of the gravid female. This
results in relatively high survival and stable transformation (Presnail and Hoy, 1992).
Maternal Microinjection
Maternal microinjection involves injection of DNA into an adult female to deliver DNA to
the eggs. The adaptation of this method to the parasitoid, Cardiochiles diaphaniae Marsh
has been reported by Presnail and Hoy (1996). The results of preliminary dissections and
injections with the plasmid pJKP2 suggested that this method could be used to deliver
injected DNA to the ovaries of the wasp, C. diaphaniae. Of the 16 females that were injected
and allowed to produce offspring, several individuals contained the plasmid sequences.
In another experiment, the plasmid phsopd, containing the parathion hydrolase gene (opd )
of Pseudomonas diminuta Leifson and Hugh, was injected. The plasmid probe hybridized to
high molecular weight DNA from three of 38 G1 adults. A fourth adult produced a hybridization
pattern consistent with integration of plasmid into the nuclear genome. The results
indicated that maternal microinjection can result in transformation of the parasitoid. The
persistence of DNA injected into two species of the adult female phytoseiid, M. occidentalis
and its transmission to serial eggs deposited by them has been assessed by polymerase
chain reaction (PCR) (Presnail and Hoy, 1994). The effect of DNA concentration on persistence
and transmission was examined in females of M. occidentalis microinjected with
plasmid DNA at three different concentrations (250, 500, 750 ng mL-1 ) and allowed to deposit
one to fi ve eggs. The plasmid DNA was found in 82% of the females assayed and in 70% of
all the eggs, including the fi fth egg produced after microinjection. Transmission of DNA
to multiple eggs was also examined in Amblyseius fi nlandicus (Oud.). The persistence and
presence of plasmid DNA in both eggs and females suggested that maternal microinjection
was a more effi cient method for DNA delivery than traditional egg microinjection, and
that it could be useful as a DNA delivery system in other phytoseiids.
Presnail et al. (1997) transformed four lines of M. occidentalis by maternal microinjection.
Putatively transformed lines were identifi ed by standard PCR in the G1 generation. After
30 generations, the lines were examined by Southern blot hybridization, and the plasmid
probe was hybridized to uncut high molecular weight DNA from all four lines, indicating
that the transgene was associated with high molecular weight DNA. In restriction-digested
Southern blots, one of the four lines displayed a hybridization pattern consistent with integration
of two plasmids into the chromosomal DNA. All four colonies were confi rmed to
be positive by PCR after 150 generations. Colonies examined for mRNA expression after
100 generations displayed PCR products consistent with transcription of the introduced
genetic sequences. Microinjection of early preblastoderm eggs in gravid females of M. occidentalis
resulted in relatively high levels of survival and transformation (Presnail and Hoy,
1992). Transformation was achieved without the aid of any transposase-producing helper
plasmid. The predatory mite was transformed with a plasmid containing the Escherichia
coli Escherich beta-galactosidase gene regulated by the Drosophila hsp70 heat-shock promoter.
Putatively transformed lines were isolated based on beta-galactosidase activity in
fi rst-generation larvae. Transformation was confi rmed in the sixth generation by PCR
amplifi cation of a region spanning the Drosophila/E. coli sequences. This method can also
be adapted to other benefi cial arthropods, particularly the phytoseiids.
Sperm-Mediated Transfer
DNA has been introduced successfully into individual honeybees by inseminating
virgin queens with linearized DNA and semen (Robinson et al., 2000), and the trait was