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Identification, distribution and ecology of malaria mosquitoes in the eastern
slopes of the Andes Mountains, Bolivia.
Gary N. Fritz1, Corey Brelsfoard1, Reema
Paudel1, Roberto Rodriguez2.
1Department of Biological Sciences, Eastern
Illinois University, Charleston IL 61920
2Laboratorio de Entomología Médica, Escuela Técnica de Salud Pública,
Cochabamba, Bolivia
Introduction
Malaria continues to be the most important vectored infectious
disease of humans, killing 1-3 million people worldwide each year. Though over a
dozen species of anopheline mosquitoes have been implicated in malaria
transmission in South America, little is known about the ecology and
distribution of most species. A lack of reliable and efficient means of
identifying malaria mosquitoes has been a major obstacle for studies on their
biology, ecology, and behavior, and responsible for our fragmentary
understanding of the epidemiology of malaria in this region of the world. This
study examined entomological aspects of the epidemiology of malaria in a
Neotropical valley system along the eastern slopes of the Andes Mountains in
Bolivia, the Chapare/Carrasco Valleys. This study had three main goals: 1) the
development of quick and reliable molecular diagnostics for identifying malaria
mosquito species, 2) the determination of species numbers and distributions in
an ecologically complex valley system in Bolivia with endemic malaria, and 3)
the relationship between aquatic breeding sites and other physical parameters in
relation to mosquito species distributions.
Methods and Results
Development of Molecular Diagnostics
By using species specific sequences from a rDNA spacer (the internal transcribed
spacer 2, Figure 1) multiplex PCR were developed for identifying all life stages
of nine species of malaria mosquitoes.
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Figure 1A. rDNA unit showing ITS2
region sequenced for alignment. |
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Figure 1B. Example alignment of the
ITS2 of four species of malaria mosquitoes showing variable regions
(black letters). |
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Figure 1C. Gel showing species
specific banding pattern for four species. |
Species Distributions and Number
Up to fifty mosquito larvae were sampled and identified (using PCR)
from fifty six aquatic breeding sites along the length of the Chapare/Carrasco valleys
(Figure 2A). The distributions of most species is non-random across all fifty
six sites
(e.g., Figure 2B). Some species such as An. trinkae, for example, are found only
at higher elevations on the eastern slopes of the valley (Figure 2B). Though
thirteen
species have been identified (Figure 3), two species, An. trinkae and An. rangeli,
comprise over fifty four percent of all mosquitoes collected.
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Figure 2A. Map of the Chapare/Carrasco valleys,
Bolivia showing fifty six breeding sites where larvae were
collected. |
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Figure 2B. example of the distribution of four of the
nine species identifiable by PCR in this study. |
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Figure 3. All species identified in the Chapare/Carrasco
valleys to date. |
Aquatic Parameters & Species Distributions
Principal component analysis and Spearman’s Correlations
indicated significant associations between
the presence/absence of particular species and particular parameters of
aquatic breeding sites
(Figures 4A and 4B). ANOVA indicated significant associations of certain species
with elevation above
sea level.
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Figure 4A. An example of Principal Component Analysis
for the presence/absence of An. trinkae at fifty six aquatic
sampling sites in
relation to fourteen aquatic parameters; |
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Figure 4B. Spearman’s Correlation Probability values
for four species of anopheline mosquitoes with respect to fourteen
water quality parameters. |
Conclusions
Multiplex PCR of nine species produce species specific amplicons useful for
identifying any life stage. The development of multiplex PCR that can diagnose
nine vectors of human Plasmodium in the Neotropics should make it more feasible
to initiate studies on anopheline mosquito ecology and the dynamics of
transmission of malaria, particularly since the primers can be used with a
minute amount of sample material for species identification.
Some species, such as An. trinkae, An. strodei, and An. marajoara exhibit narrow
distribution ranges, whereas others species such as An. triannulatus and
An. rangeli are broadly distributed. At least thirteen species have been identified in the Chapare/Carrasco valleys, but two species,
An., trinake and An. rangeli comprise over fifty four percent of all specimens collected Distribution patterns of some species were
significantly associated with certain water quality parameters (e.g., dissolved
solids, pH, conductivity, etc.), elevation, aquatic categories (river, pond,
ditch, etc.) and their associated flora (algae, grasses, etc.).
Acknowledgements
We thank the Bolivian Ministerio de Salúd, the Cochabamba Ministerio de Salúd,
the Laboratorio de Entomología Médica at the Escuela Técnica de Salúd in
Cochabamba, Dr. Rene Mollinedo, Claudia Lopez, Dr. Hernan Bermudez, CUMETROP,
Escuela de Medicina de la Universidad de San Simón. This project was supported
by the Council for Faculty Research, EIU and NIH Grant #1 R15 AI47796-01A1. Also
this project was supported by a Charles B. Arzeni Tropical Biology Scholarship.
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