Biology Reference
In-Depth Information
FIGURE 6.1
The actinomycete symbiont
Rhodoccocus rhodnii
lives in the gut of
Rhodnius prolixus
, in direct
proximity to the ChagasÔ disease agent
Trypanosoma cruzi
. [From Beard, C.B., Durvasula, R.V., and Richards,
F.F. (2000). In
Insect Transgenesis: Methods and Applications
(A.M. Handler and A.A. James, Eds.), pp. 289Ï303.
CRC Press, Boca Raton, FL. With permission.]
are well described (Beard et al., 1993; Beard
and Aksoy, 1997). We have developed a series of shuttle plasmids that maintain low copy numbers
in
Methods for genetic manipulation of
R. rhodnii
transformed to express resistance to the antibiotic
thiostrepton were introduced to Ýrst-instar aposymbiotic
R. rhodnii
. In initial studies,
R. rhodnii
. These bacteria were maintained
in the host bugs throughout sexual maturation for a period of 6.5 months (Beard et al., 1992). Assay
of the host
R. prolixus
at all developmental
stages. No adverse effects of the genetically altered bacteria on growth and fecundity of the bugs
were noted. This study established the principle of paratransgenic expression of foreign genetic
material in an arthropod vector.
R. prolixus
revealed thiostrepton-resistant
Rhodococcus rhodnii
CECROPIN A EXPRESSION IN PARATRANSGENIC
RHODNIUS PROLIXUS
The search for antitrypanosomal molecules that could be used in a paratransgenic strategy led to
studies involving the immune peptide
-cecropin A. Cecropin A, isolated from the moth
Hyalophora
L
cecropia
, is a member of a family of nonspeciÝc peptides present in insects and certain vertebrates.
It is a pore-forming peptide that has a broad spectrum of antibacterial activity and plays an important
role in insect innate immunity (Boman, 1991). We demonstrated paracidal activity of cecropin A
against several strains of
(
Table 6.1)
.
We then cloned cDNA for cecropin A in the shuttle plasmid pRrThioCec (
Figure 6.2)
and
established a line of cecropin AÏproducing
T. cruzi
, with virtually no activity against
Rhodococcus rhodnii
R. rhodnii
(Durvasula et al., 1997).
An experimental colony of aposymbiotic
Rhodnius prolixus
was fed cecropin-producing
Rhodo-
coccus rhodnii
and challenged with strain DM28
T. cruzi
. A control group of
Rhodnius prolixus
was fed wild-type
Rhodococcus rhodnii
and subjected to the same
T. cruzi
challenge. These studies
revealed clearance of
T. cruzi
in 65% of experimental group
Rhodnius prolixus
, with a 2 to 3 log
reduction of parasite count in the remaining 35%. Control group
R. prolixus
supported
T. cruzi
at
concentrations of 10
parasites/bug. Results shown in
Figure 6.3
are from the Ýrst experiment;
several repeat trials involving 100 insects in each group conÝrmed our initial Ýndings.
The transformed
to 10
5
6
in these studies served normal symbiotic functions in
the host bugs. Growth rates and fecundity of paratransgenic
Rhodococcus rhodnii
equaled those of
wild-type bugs. Furthermore, expression of cecropin A and resistance to thiostrepton persisted
throughout the 7-month duration of the study in the absence of antibiotic selection. The pRrThioCec
Rhodnius prolixus
