Biology Reference
In-Depth Information
A SPREADING STRATEGY FOR POTENTIAL FIELD APPLICATION
Field use of the paratransgenic approach to control of ChagasÔ disease requires a strategy for
delivery and spread of transgenic
R. rhodnii
among natural populations of
Rhodnius prolixus
. Modes
of delivery of engineered bacteria to vector populations must simulate naturally occurring methods
of symbiont spread. An optimal spreading strategy would involve minimum numbers of genetically
altered organisms with maximum uptake and activity of the transgenic material. The transgenic
symbionts should be competitive with wild-type Þora to establish predominant infections in the
target vector populations. Furthermore, spread of foreign genes via engineered bacteria should
occur only in targeted populations of vectors, with limited uptake by nontarget arthropods or other
environmental bacteria. In the following sections we review our ongoing and proposed studies
aimed at development of a robust spreading mechanism for engineered
Rhodococcus rhodnii
.
CRUZIGARD: A SUBSTRATE FOR GENE DELIVERY
Spread of
R. rhodnii
within populations of
Rhodnius prolixus
occurs via coprophagy, the ingestion
of fecal deposits. Newly emerging Ýrst-instar nymphs are transiently aposymbiotic (devoid of
symbiotic bacteria). Probing of fecal droplets deposited by adult bugs either on the eggshell or in
the immediate environment permits Ýrst instar nymphs to ingest
R. rhodnii
and establish gut
infection. Nymphs reared in sterile chambers from surface-sterilized eggs fail to mature beyond
the second instar stage. Delivery of
R. rhodnii
via membrane feeder to aposymbiotic nymphs by
the second molt permits normal development and sexual maturation.
We have developed a simulated fecal paste Ð termed CRUZIGARD Ð that permits delivery
of
R. rhodnii
to colonies of
Rhodnius prolixus
. CRUZIGARD is comprised of an inert guar gum
matrix impregnated with 10
8
CFU/ml of
Rhodococcus rhodnii
. India ink is added to achieve the
black color of natural
Rhodnius
feces. Survival of
R. rhodnii
in this form is between 6 and 8 weeks.
Initial studies of CRUZIGARD involved aposymbiotic colonies of
Rhodnius prolixus
. Apo-
symbiotic Ýrst-instar nymphs exposed to CRUZIGARD containing genetically altered
Rhodococcus
rhodnii
reached sexual maturity at a rate comparable to similar nymphs exposed to natural feces.
Sampling of adult bugs in this study revealed antibiotic resistance markers indicative of genetically
altered
R. rhodnii
. These initial studies conÝrmed that CRUZIGARD could approximate natural
mechanisms for delivery of genetically altered bacteria without causing excess toxicity to the bugs.
However, these studies were performed in very conÝned spaces under sterile conditions. Field
populations of
Rhodnius prolixus
and other reduviid bugs may harbor over 20 different types of
bacteria and fungi (P. Pennington, unpublished data). Competition from these microbes is an
important determinant of the ultimate success of the CRUZIGARD strategy.
To address issues of bacterial competition, a study was conducted at the Medical Entomology
Research and Training Unit/Guatemala (MERTU/G) (Durvasula et al., 1999b). In this trial, our
goal was to assess the efÝcacy of CRUZIGARD under simulated Ýeld conditions. Insects and cage
materials for this study were collected from the ChagasÔ diseaseÏendemic region of Olopa in Eastern
Guatemala. Lucite cages (0.6 0.6 0.6 m) were constructed and lined with dirt. Panels of thatch
and adobe measuring 20 20 cm were made and impregnated with CRUZIGARD to cover 50%
of the surface area
(Figure 6.6).
Rhodococcus rhodnii
transformed to express DB3 and kanamycin
resistance were used in the experimental cage; wild-type
R. rhodnii
were applied to the control
cage. Eight adult male and female
Rhodnius prolixus
were placed in each cage until egg laying
was complete. Fecal droplets deposited by these adults remained in the cages, providing bacterial
competition to the genetically altered bacteria in CRUZIGARD.
Sampling of instar nymphs at the fourth, Ýfth, and adult stages revealed that approximately
50% of bugs carried kanamycin-resistant
Rhodococcus rhodnii
, conÝrmed by both culture and
polymerase chain reaction. Other environmental microbes, such as
Staphylococcus
spp.,
Pseudomo-
nas
spp., and Candida, were isolated from the bugs. Bugs that harbored recombinant
R. rhodnii
