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Use of the pupal/demographic-survey technique to identify
the epidemiologically important types of containers
producing Aedes aegypti (L.) in a dengue-endemic area of
Venezuela
A. E. LENHART
*
, C. E. CASTILLO
{
, M. OVIEDO
{
and E. VILLEGAS
{
*
Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, U.K
{
Centro de Investigaciones ‘Jose´ Witremundo Torrealba’, Universidad de los Andes, Nu´cleo
Universitario ‘Rafael Rangel’, Trujillo, Venezuela
Received 23 January 2006,
Accepted 27 January 2006
As dengue continues to emerge as a major public-health problem world-wide, efforts to control the dengue vector
Aedes aegypti must become more effective and efficient. Results from larval and pupal surveys applied in Venezuela
illustrate the uniqueness of the information gained from pupal surveys; information that is lost when traditional
Stegomyia indices are calculated. As most Ae. aegypti pupae will emerge to become adults, controlling the containers
that produce the most pupae could have the greatest impact on the adult population. Pupal-survey results in
Venezuela showed that large (150- to 200-litre) water drums produce the greatest number of pupae throughout the
year. In the rainy season, approximately 70% of all pupae are found in these drums or in tyres, buckets and tanks.
Over 80% of pupae in the dry season are found in drums and tanks alone. By targeting only those domestic
breeding containers that produce the greatest number of pupae, control efforts may be streamlined to have the
greatest impact on reducing the local adult Ae. aegypti population.
Dengue is the most common and wide-
spread arboviral disease of humans world-
wide (Mairuhu et al., 1997). In the absence
of a vaccine, control of the peridomestic
vector mosquito, Aedes aegypti (L.), is the
only effective preventive measure.
Unfortunately, traditional Stegomyia indices
do not provide a complete picture of the
‘key’ containers used by Ae. aegypti as
breeding sites (i.e. those that are most
important to the production of adult Ae.
aegypti in a particular setting; Focks, 2003).
As most (.80%) of the pupae of this species
probably survive to become adults (Focks
et al., 1993), pupal surveys would appear to
be a reasonable method of identifying the
types of container that are responsible for
producing the bulk of a local population of
adult mosquitoes. Such identification of
the key container types should allow
control efforts to be better focussed and
more cost-effective.
The main objectives of the present study,
which was based in the Venezuelan city of
Trujillo, were to assess the consistency and
practicality of using pupal/demographic
surveys to identify the containers that are
the most productive breeding sites for the
local Ae. aegypti population.
MATERIALS AND METHODS
In a longitudinal study, four cross-sectional
surveys were performed in the neighbour-
hood of Tres Esquinas, in the city of
Trujillo, Ve nezuela. Trujillo City (9u229 N,
70u269 W) lies in north–western Venezuela
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E-mail: alenhart@liv.ac.uk; fax: z44 (0)151 705 3369.
Annals of Tropical Medicine & Parasitology, Vol. 100, Supplement No. 1, S1–S7 (2006)
# 2006 The Liverpool School of Tropical Medicine
at 800 m above sea level, has a population
of approximately 53,000, is the capital of
Trujillo state, has a mean annua l rainfall of
750 mm, and has air temperatures ranging
from 16–37uC. There are normally two
rainy seasons in this area — the first peaking
in April/May and the second in November
— and transmission of dengue, although
perennial, tends to peak as each rainy season
ends. The first cross-sectional survey, in
April 2004, was timed to coincided with the
beginning of the first period of dengue
transmission in 2004. To obtain an under-
standing of the most productive container
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FIG. 1. The relative importance of each category of domestic water-holding container, expressed as the
percentages of the containers encountered that were ‘wet’, holding any immature stage of Aedes aegypti, or holding
Ae. aegypti pupae, and as the percentages of the total number of Ae. aegypti pupae produced.
S2 LENHART ET AL.
habitats throughout the year, at times
of varying rainfall and dengue-
transmission risk, further surveys were con-
ducted in May, June and October of the
same year.
The neighbourhood of Tres Esquinas
consists of approximately 1000 households.
Each house was mapped using a handheld
global-positioning system (Magellan
MeridianH Gold; Thales Navigation, Santa
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Aedes aegypti PUPAL SURVEYS IN VENEZUELA S3
Clara, CA) and data were collected on all
the water-holding (‘wet’) containers in the
study area. A detailed survey that included
the collection of data on container type,
volume, location and use was conducted at
household level. At the same time, the
number of residents in each sampled house-
hold was recorded, so that the size of the
human population of the neighbourhood
could be estimated, and the results of the
pupal survey could be expressed as the
number of Ae. aegypti pupae/person — a
statistic that can help to estimate the levels
of reduction necessary to limit transmission.
In the first survey, significant efforts were
made to survey every house in the commu-
nity and 1031 houses were studied. In
subsequent surveys, fewer households parti-
cipated (774 in May, 737 in June, and 787
in October), as time constraints did not
permit repeated visits to the houses where
no-one was home when the survey team
arrived. Each wet container detected was
checked for the immature stages of mosqui-
toes. Every pupa seen was counted, col-
lected (using pipettes, ladles and fine-mesh
colanders), taken back to the laboratory,
and allowed to develop into an adult, which
was sexed and identified to species.
The data collection was carried out by 10
trained entomology technicians and over-
seen by a research entomologist and three
other senior researchers. A data-entry tech-
nician entered all the field results into an
Access (Microsoft) database.
RESULTS
In all four surveys, more pupae were
encountered in large cylindrical water
drums (with capacities of 150–200 litres)
than in any other type of container (Fig. 1).
This trend was most marked in the June
survey, when 55% of all the p upae collected
came from such drums. The percentage of
all collected pupae coming from large plastic
water tanks (found both at ground level and
on the roofs of hou ses) was also much
higher in the June survey (31.2%) than in
the three other surveys. June coincided with
the beginning of the first dry season, during
which piped water is typically rationed and
people begin to store large quantities of
water, most commonly in drums and tanks.
During wetter times of the year, when the
local residents are less inclined to store large
quantities of domestic water, other types of
wet container increase in their relative
abundance and become more important in
terms of pupal production (Fig. 1). Nearly
70% of the pupae collected during a rainy
season (i.e. in the April, May and October
surveys) came from large buckets, tyres,
tanks or drums.
For each category of container, the mean
number of pupae/positive container varied
over time (Fig. 2). Most notably, the mean
number of pupae in each positive water tank
more than doubled between May (28.2) and
June (59.0), highlighting the importance of
this type of water-storage container during
dry conditions. Over the entire study period,
only two containers (both 150- to 200-litre
drums) were found with .500 pupae each
— one with 538 pupae in the June survey
and one with 589 in the October survey.
These might both be considered atypical
infestations, as the next most productive
container, a water drum seen in the October
survey, only contained 171 pupae.
The results of the pupal survey were
compared with those of a traditional con-
tainer survey (which measures the presence
or absence of any stage of immature Ae.
aegypti in all water-holding containers),
taking into account the types of containers
that were positive. Although the results of
the traditional survey indicated that bottles
were the container type most commonly
infested with immature Ae. aegypti, the
results of the pupal survey showed that the
contribution of bottles to overall pupal
production was negligible (Fig. 3).
The data collected in the cross-sectional
surveys were used to calculate the mean
number of Ae. aegypti pupae/person in
each survey. Overall, these mean values
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S4 LENHART ET AL.
correlated only marginally well with the
traditional Stegomyia indices, corresponding
best with the Breteau index and poorest with
the container index (see Table).
DISCUSSION
The present results indicate that the meth-
odology of the pupal/demographic survey
provides a useful framework for prioritising
vector-control interventions, while high-
lighting the limitations of data collected in
traditional Stegomyia surveys. In the present
study, counting the total number of pupae
in each container offered the maximum
information for identifying the key types of
container to target in campaigns for the
control of Ae. aegypti, and so achieving
maximum impact. In Trujillo, dengue
transmission is year-round but peaks occur
soon after periods of heavy rainfall. Since
transmission is perennial, the year-round
targeting of the more productive containers,
with larviciding or source reduction, is
recommended. By comparing the results of
the pupal/demographic surveys with esti-
mates of the corresponding traditional
Stegomyia indices, it became apparent how
the latter may greatly distort the true
importance of certain container types in
the production of Ae. aegypti pupae and,
ultimately, adults.
Computing the numbers of pupae/person,
instead of using the traditional indices that
are based on all immature stages, gives a
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FIG. 2. Seasonal variation of pupal productivity by container type.
TABLE. The levels of correlation [shown as Pearson’s
correlation coefficients (r)] between the pupae/person index
(PPI), in each of the four cross-sectional surveys, and the
corresponding values for the traditional Stegomyia indices
Correlate with PPI r
PPI 1
Premises index
*
0.87
Container index
{
0.25
Breteau index
{
0.93
*
Percentage of premises with immature stages of Ae.
aegypti.
{
Percentage of wet containers with immature stages of
Ae. aegypti.
{
Number of containers with immature stages of Ae.
aegypti/100 premises.
Aedes aegypti PUPAL SURVEYS IN VENEZUELA S5
unique measure of infestation, which could
be potentially useful in assessing the risk of
dengue transmission in a particular setting
at a given time.
In addressing the question of consistency
of the pupal-survey results over time, it
became apparent that water drums consis-
tently produced more pupae than any other
category of container. A year-round inter-
vention targeted at such drums is therefore
recommended. Other interventions could be
tailored to the season. The recommended
containers to target during the rainy season
(in addition to drums) would be large
buckets, tanks and tyres; these are all easily
identifiable containers which, with the
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FIG. 3. The relative importance of each container type to the production of Aedes aegypti may be perceived
differently according to which survey technique is used. Here, the data from the April 2004 survey are presented in
three ways: as the percentages of the containers found to harbour any immature stage (a) or the pupae (b) of Ae.
aegypti, and as the relative contribution of each container type to the local production of Ae. aegypti pupae (c).
Although the counts of containers with any immature stages (a) indicated that bottles were by far the most
important category of container, bottles contributed only slightly to pupal production (c).
S6 LENHART ET AL.
drums, produced approximately 70% of the
pupae during the rainy season. A dry-season
intervention could be focussed only on
drums and water tanks — the two container
types that together produced .80% of dry-
season pupae. With the exception of tyres,
nearly all of the most highly productive
containers encountered in the present study
were being used to store domestic water.
In terms of their practicality, the pupal/
demographic surveys, while initially tedious,
can potentially reduce the costs of interven-
tion activities and make them quicker.
Although it requires patience and precision
to search for pupae and count every one
found, interventions can be more efficiently
targeted once a picture of the most produc-
tive containers emerges. An intervention
targeted only at water drums (which, in
the case of Trujillo only represented 3%–
19% of all water-holding containers), for
example, would require significantly less
effort than an unfocussed intervention that
was designed to treat or eradicate all water-
holding cont ainers. The most productive
containers need only be identified once, just
before beginning an interventio n.
It remains unknown if, by attacking only
the more productive types of container, the
overall pupal (and subsequently adult)
population of Ae. aegypti will significantly
decrease. Further research will help to
answer this question, as well as to clarify
the relationships between pupal production,
the adult population and, ultimately, den-
gue transmission.
ACKNOWLEDGEMENTS. The authors are
deeply grateful to the field team who
carried out the surveys and to the people
of the study neighbourhood, for their
collaboration. This research was funded by
a grant from the UNICEF/UNDP/WORLD
BANK/WHO Special Programme for Research
and Training in Tropic al Diseases.
REFERENCES
Focks, D. A. (2003). A Review of Entomological
Sampling Methods and Indicators for Dengue Vectors.
Document TDR/IDE/Den/03.1. Geneva: World
Health Organization
Focks, D. A., Haile, D. G., Daniels, E. & Mount, G. A.
(1993). Dynamic life table model for Aedes aegypti
(Diptera: Culicidae): analysis of the literature and
model development. Journal of Medical Entomology,
30, 1003–1017.
Mairuhu, A. T. A., Wagenaar, J., Brandjes, D. P. M. &
van Gorp, E. C. M. (2004). Dengue: an arthropod-
borne disease of global importance. European Journal
of Clinical Microbiology and Infectious Diseases, 23,
425–433.
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