Biology Reference
In-Depth Information
6.
Genetic manipulation of symbiotic bacteria should not render them virulent, either to
the target vector or other organisms in the environment. Furthermore, bacteria chosen as
gene-delivery vehicles must not be pathogens themselves.
7.
A robust method must exist for delivery of genetically altered symbionts within Ýeld
populations of the disease-transmitting arthropods. Ideally, this method would mimic
naturally occurring mechanisms for symbiont dispersal and would not result in increased
populations of the disease-transmitting vectors. Strategies of foreign gene dispersal
should target appropriate vectors selectively and minimize nontarget uptake and retention
of recombinant genetic material. Methods for assessing environmental spread of foreign
DNA and prediction of limits of spread must exist as part of a paratransgenic approach.
In summary, paratransgenic manipulation of disease-transmitting vectors requires critical evalu-
ation of the natural biology of vector populations. Careful assessment of patterns of symbiont trans-
mission, population dynamics, reproductive strategies, and vector interactions with the environment
is the cornerstone for the development of a paratransgenic approach that mimics natural phenomena.
THE TRIATOMINE BUG
RHODNIUS PROLIXUS
:
A PARADIGM FOR PARATRANSGENESIS
Though the paratransgenic strategy may be applied to a variety of disease-transmitting arthropods,
it has been most successfully developed in the reduviid bug,
. A member of the
Triatominae, this bug is widely distributed in Central America and northern regions of South
America. It is an important vector of
Rhodnius prolixus
, the causative agent of ChagasÔ disease.
Over 50,000 people die annually of this disease, and nearly 90 million individuals are at risk for
this disease due to exposure to the reduviid vectors. Neither a cure nor a vaccine exists for ChagasÔ
disease. Control of transmission has hinged largely on insecticide campaigns aimed at elimination
of reduviid vectors. These have been quite successful in areas of the Southern Cone countries, but
issues of cost, maintenance, environmental toxicity, and vector resistance remain.
The symbiotic relationships between triatomine bugs and soil-associated actinomycete bacteria are
well described.
Trypanosoma cruzi
R. prolixus
maintains a close relationship with the actinomycete
Rhodococcus rhodnii
.
The bacterium aids in the processing of B-complex vitamins found in the diet of
Rhodnius prolixus
and
aids in sexual maturation of the bug.
, which are reared from surface-sterilized eggs in sterile
laboratory chambers, fail to develop beyond the second molt. Delivery of the bacteria to aposymbiotic
nymphs via blood meals across a membrane restores normal growth and fecundity of the nymphs.
R. prolixus
Rhodococcus rhodnii
exists as an extracellular, intraluminal symbiont in the hindgut of
Rhod-
nius prolixus
colony-forming units (CFU)/ml. It is
juxtaposed in this environment with the infective trypomastigote form of
where it may reach concentrations of 10
8
T. cruzi
(
Figure 6.1)
.
The
amenability of
Rhodococcus rhodnii
to isolation and genetic manipulation and its proximity to the
pathogen
in the bug gut satisÝes several requirements for paratransgenic manipulation of
the host reduviid bug.
T. cruzi
PARATRANSGENIC MANIPULATION OF
RHODNIUS PROLIXUS
Isolation and culture of
Rhodococcus rhodnii
from both laboratory and Ýeld-caught
Rhodnius
prolixus
is quite simple. Fecal extracts of bug hindgut contents are suspended in physiologic
saline and plated aerobically on brain heart infusion agar at 28
appear
within 3 to 5 days as discrete, rough colonies of a pink-white hue. Older colonies exhibit heaped
or ÑvolcanicÒ growth patterns characteristic of several actinomycetes. In liquid BHI medium
under conditions of vigorous agitation (200 to 225 rpm),
∞
C.
Rhodococcus rhodnii
R. rhodnii
reach concentrations of 10
8
to 10
CFU/ml with a generation time of approximately 18 h.
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