The adult mosquito bioassys currently employed
by MRCU have been developed by the Centers for Disease Control and Prevention,
Atlanta. The bioassay test they have developed is intended to be simple
to perform so that mosquito control operators can carry out resistance
testing with the need for expensive laboratory equipment.
The bottle bioassay test is performed by introducing live adult mosquitoes
into a glass bottle that has been coated with a known amount of a certain
types of insecticide. Once the mosquitoes are inside the bottle the time
taken for all mosquitoes to die is recorded. From this a time / mortality
curve can be produced. By comparing time / mortality curves with mosquitoes
from different locations, or the same locations over time, an assessment
of the development of resistance can be made.
Time mortality data gathered from the bottle bioassays is a measure of
the time it takes for a compound to penetrate the mosquito, traverse intervening
tissues, get to the target site and act on the target site. Anything that
prevents or delays the compound from doing its job is contributing to
resistance. Bottle Bioassays have been conducted on mosquitoes collected
from a number of different sites on Grand Cayman against a range of different
insecticides.
The samples of mosquitoes tested so far have not shown resistance to
Permethrin, although when combined with Piperonyl butoxide better results
are observed. It can be concluded that Permethrin is an effective active
ingredient for use against adult Aedes taeniorhynchus particularly when
combined with Piperonyl butoxide. The results for naled (Dibrom) have
also been promising. Prior to using bottle bioassays it was believed that
resistance to this active ingredient was widespread in Grand Cayman. There
is currently no evidence to suggest reduced susceptibility to naled (Dibrom).
Microplate assays have also been developed
by CDC, Atlanta to help determine whether enzymes are present in the mosquitoes
that can cause insecticide resistance. A
number of relatively simple tests can be used to measure Non Specific
Esterase, Haeme Peroxidase, Glutathione-S-Transferase, Acetylcholine Esterase
and Insensitive AcetylCholine esterase levels. During
1998 MRCU conducted assays of non-specific esterase, haeme peroxidase
as well as some nonspecific Acteylcholine esterase enzyme. It
is proposed to expand this testing to all of the enzymes listed during
1999.
Enzyme Assay Results:
Initially only two enzyme assays have been performed (heme-peroxidase
and nonspecific oxidase).
Later insensitive AcetylCholine Esterase Assay was performed on mosquitoes
from a single location.
1. Nonspecific Esterase
Above figure shows values for nonspecific Esterase assays performed on
adult mosquitoes collected from South Sound, Canal Point, Red Bay and
Jackson's Pond. Data from CDC Atlanta has indicated that Absorbance values
greater than 0.9 represent the presence of elevated esterase enzymes.
These results show that Mosquitoes sampled from all locations have a proportion
of the sample with values greater than 0.9. Elevated levels of esterase
enzymes are often associated with the development of resistance to Organophosphate
insecticides, although different esterase enzymes may confer resistance
to different insecticides within the same group.
2. Heme-peroxidase
Above figure shows values for heme-peroxidase assays carried out on adult
mosquitoes collected from South Sound, Canal Point, Red Bay and Jackson's
Pond. As with Nonspecific Esterase results a value of greater than 0.9
is usually indicative of elevated oxidase levels. The results obtained
so far do not indicate the presence of elevated oxidase enzymes, which
are usually associated with the development of resistance to Pyrethroid
insecticides.
3. Insensitive AcetylCholine esterase
Above figure shows insensitive acetylcholine esterase assays for adult
mosquitoes collected from Jackson's Pond. In mosquitoes demonstrating
insecticide resistance through this mode of action, a significant difference
in the values for T0 and T10 would be expected. The results obtained so
far do not demonstrate the presence of insensitive acetylcholine esterase.
