Practical Manual For Dengue Surveillance-MRI

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Chapter 1
1. Introduction
Dengue and Dengue Haemorrahgic Fever (DHF) is caused by one of four closely related but
antigenically distinct virus serotypes (DEN 1, DEN 2, DEN 3, and DEN 4) of the genus Flavivirus .
The disease is primarily an urban disease of the tropics, and the viruses are maintained in a cycle that
involves humans and Aedes (Stegomyia) mosquitoes. Over the past, two decades there has been a
dramatic global increase in the frequency of Dengue Fever (DF), Dengue Haemorrhagic Fever (DHF) and
Dengue Shock Syndrome (DSS) and their epidemics.
1.1 Origin and Natural history

The origin of the dengue viruses is said to have an African origin and distribution around the
world with the slave trade (Hirsch, 1883, Smith 1956) or the viruses may have originated in a forest cycle
involving lower primates and canopy dwelling mosquitoes in the Malay Peninsula (Smith 1956, Rudnick
and Lim 1986). Regardless of the geographic origin, the dengue viruses most likely evolved as viruses of
mosquitoes before becoming adapted to lower primates and humans. Biologically dengue viruses are
highly adapted to their mosquito host and are maintained in mosquito species responsible for forest
cycles, with periodic amplification in lower primates (Rudnick and Lim 1986, Cornet 1993). It is possible
that different virus serotypes evolved in taxonomically related mosquito species in different geographical
regions. All four virus serotypes have been documented in forest cycle in Asia while only one (DEN 2)
has been documented in Africa (, Rudnick and Lim 1986, Cornet 1993). It is currently thought that these
viruses probably had an Asian origin which is supported by sero surveys conducted in rural communities
of Malaysia in the early 1950s (Smith 1956). At some point in the past, probably with the clearing of the
forests and development of human settlements, dengue viruses moved out of the jungle and into a rural
environment where they were and still are transmitted to humans by peri domestic mosquitoes such as Ae.
albopictus. Migration of people and commerce ultimately moved the viruses into the villages, towns and
cities of tropical Asia where the viruses were most likely transmitted sporadically by Ae.albopictus and
other closely related peri domestic Stegomyia species (Gubler 1997).
It has been suggested that the principal epidemic vector of dengue viruses, Ae. aegypti was a
New World species. As pointed out by Dyar (1928), Carter (1931) and Christopher (1960), however,
Ae.aegypti is most likely of an African origin for the following reasons.
1. There are no closely related Stegomyia species in the Americas where there are numerous such
species of the same sub genus in both the Ethiopian and Oriental regions.
2. Ae. aegypti occurs in Africa as a widespread feral species, breeding in the forest independent of
humans. Although occasionally found occupying natural larval habitats in Asia and Americas, it
is primarily an urban species in both of these regions and only rarely occurs in the absence of
man.
Medical Research Institute and Dengue coordination Unit - 2011

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Therefore current thinking is that Ae. aegypti had an African origin and had adapted to the peri
domestic environment, breeding in water storage containers in African villages prior to the slave trade.
The latter provided the mechanism for the species to be introduced to the New World. The species
become closely adapted to humans and was a common passenger on sailing vessels during the 17th, 18th
and 19th centuries. Thus by 1800, Ae aegypti had already become established in many large tropical cities
around the world specially in Africa and the New World. In Asia there is evidence that Ae. aegypti never
became the predominant Stegomyia species in many non coastal cities until during and after World War II
(Smith 1956).
During the war, existing water systems were destroyed and water storage was increased for
domestic use as well as for fire control. War equipments were moved between cities and countries and
large amounts of equipments were also left behind. This material collected rain water and made ideal
larval habitats for Ae. aegypti , resulting in the transport of mosquitoes and their eggs to new geographical
areas. The result of these ecological changes was a greatly expanded geographical distribution and
increased population densities of Ae. aegypti. In addition many thousands of Japanese and allied soldiers,
most of them susceptible to dengue virus infection, were constantly moving between countries in Asia
and the Pacific. This provided ideal conditions for movement of viruses between cities, countries and
other regions as well as susceptible individuals for epidemic transmission. The war years were thus
responsible for creating the conditions for the emergence of DHF in South East Asia.
In the years following World War II unprecedented urbanization of South East Asia began with
millions of people moving to the cities of the region. Urban centres in most countries expanded rapidly in
an uncontrolled and unplanned fashion. Housing was inadequate, and water sewer and waste management
systems deteriorated. The Ae. aegypti populations and dengue viruses thrived in this new ecological
setting, with increased transmission and increased frequency of dengue epidemics occurring in the
indigenous populations of children. Moreover, economic expansion began in the region that continues
even today. This led to continued urbanization and increased movement of people between cities and
countries. Those countries that did not already have multiple virus sero types co-circulating quickly
became hyper endemic. The viruses often all four sero types were maintained in a human Ae. aegypti
human cycle in most urban centres of South East Asia (Gubler 1997). In addition to Ae. aegypti and Ae.
albopictus , some other Aedes species belong to the Stegomyia and Finlaya sub groups have been
incriminated as vectors.
Dengue epidemics have known to occur over the last three centuries in tropical, sub tropical and
temperate areas of the world. In every country where the disease emerged as a major public health
problem, it evolved in a similar manner, first as sporadic cases of DHF occurring for several years,
ultimately cumulating in a major epidemic. Following the first epidemic, a pattern of epidemic activity
was established, with epidemics occurring every 3to 5 years. Characteristically, succeeding epidemics
became progressively larger as a result of geographical expansion of DHF within the country.

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Fig. 1.1 Countries/Areas at risk of dengue transmission (WHO 2008)
Fig 1.2 Known global distribution of the four dengue virus serotypes showing areas of
hyperendemicity

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The first epidemic of dengue was recorded in 1635 in the French West Indies although a disease
compatible with dengue had been reported in China as early as 992 AD. The disease was called water
poison by the Chinese and was thought to be somehow connected with flying insects associated with
water. Thus dengue or very similar illness had a wide geographical distribution before 18th century, when
the first known pandemic of dengue like illness began. First recorded outbreak of dengue disease
compatible with DHF occurred in Australia in1897. A similar haemorrhagic disease was recorded in
1928 during an epidemic in Greece and again in Taiwan in 1931. The first confirmed epidemic of DHF
was recorded in the Philippines in 1953 1954. Since then major outbreaks of DHF with significant
mortality have occurred in most countries of the South East Asia Region including India, Indonesia,
Maldives, Myanmar, Sri Lanka and Thailand and also in the Western Pacific Region (Norman Gratz
1997)
In 1998 this mosquito borne disease is the most important tropical infectious disease after
malaria, with an estimated 100 million cases of dengue fever, 500,000 cases of dengue haemorrhagic
fever and 25,000 deaths annually. The reason for this resurgence and emergence of dengue haemorrhagic
fever in the waning years of 20th century are complex and not fully understood, but demographic, societal
and public health infrastructure changes in the past 30 years have contributed greatly.
1.2 Dengue situation in Sri Lanka
In Sri Lanka clinical dengue like illness has been recorded since the beginning of the 20th century
and it was serologically confirmed in 1962. There was an island wide epidemic of dengue associated with
DEN 1 and DEN 2 with 51 cases of DHF and 15 deaths in the period of 1965 1968. From 1969 to
1988, multiple dengue serotypes circulated in urban areas with endemic DF, but there were only
occasional cases of DHF.
DHF has become endemic in Sri Lanka from 1989 onwards and there have been 203 hospitalized
clinical cases of DHF and 20 deaths. In 1989 DHF cases initially occurred mainly in and around Colombo
but they progressively spread to other towns and reached outbreak proportions in several provincial
capitals e.g. Kurunegala, Kandy, Batticaloa in 1996 (Tissa Vitarana 1997). Since then a dramatic increase
in the incidence of dengue and its severe manifestations have been observed in the country.
In 2002 there were 8931 cases and 64 deaths followed by 15434 cases and 88 deaths in 2004.
The highest number of cases were reported in 2009 with 35007 cases and 346 deaths ( Fig 1.3) . At
present dengue fever and dengue haemorrhagic Fever (DF/DHF) show a high endemic pattern in Sri-
Lanka. The percentage prevalence of dengue cases in different provinces in the country is shown in fig
1.4 (Epidemiology Unit, Ministry of Health, Sri Lanka).

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Fig. 1.3
Dengue cases and deaths in Sri lnka from 2000 - 2010
40000 400
35000 350
30000 300
25000 250
20000 200
15000 150
10000 100
5000 50
0 0
2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
cases deaths
Fig. 1.4
Provincial distribution of Dengue cases in Sri Lanka 2010
Eastern
Uva 10%
western
7% 33%
Sabaragamuwa
11%
North central
5%
central
North Western 7%
Southern Nothern
7%
7% 13%

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1.3 Seasonal Distribution
Seasonal variation in population density and distribution is common for Ae. aegypti since it is
sensitive to changes in temperature, rainfall, humidity, latitude, and altitude. Eventually low mosquito
populations are evident in dry and cool seasons and they increase when temperatures increases and the
wet season commences (Schultz 1993). Biological and entomological parameters related to a seasonal
pattern of dengue using mathematical model reported that the strongest influence on the seasonality and
pattern of dengue transmission is the duration of infectiousness of the host, vector mortality and biting
rates (Bartley et al 2002). Susceptibility to dengue viruses among Ae.aegypti mosquitoes collected in
different seasons of the year showed no seasonal correlation even though a seasonal pattern of dengue
transmission was observed in Thailand. It was suggested that characteristics of the virus, vector density
and frequency of host-vector contact should also be considered (Thongrungkiat et al 2003).
1.4 The virus
Dengue virus (DEN) is a small single-stranded RNA virus comprising four distinct serotypes
(DEN-1 to -4). These closely related serotypes of the dengue virus belong to the genus Flavivirus, family
Flaviviridae. The mature particle of the dengue virus is spherical with a diameter of 50nm containing
multiple copies of the three structural proteins, a host-derived membrane bi-layer and a single copy of a
positive-sense, single-stranded RNA genome. The genome is cleaved by host and viral proteases in three
structural proteins (capsid, C, prM, the precursor of membrane, M, protein and envelope, E) and seven
nonstructural proteins (NS).
Distinct genotypes or lineages (viruses highly related in nucleotide sequence) have been identified within
each serotype, highlighting the extensive genetic variability of the dengue serotypes. Purifying selection
appears to be a dominant theme in dengue viral evolution, however, such that only viruses that are fit
for both human and vector are maintained. Among them, Asian genotypes of DEN-2 and DEN-3 are
frequently associated with severe disease accompanying secondary dengue infections (Leitmeyer KC.
1999, Lanciotti RS et al 1994, Messer WB. 2003 ). Intra-host viral diversity has also been described in
human hosts.
1.5 The host
Humans are the main amplifying host of the virus. Human get the virus by the bite of an infected
Aedes mosquito. After a person is bitten by an infective mosquito, the virus undergoes an incubation
period of 3 to 14 days ( average 4 to 7 days) after which the person may experience acute onset of fever
accompanied by a variety of nonspecific signs and symptoms(Siler et al 1926). During this acute febrile
period, which may be as short as two days and as long as 10 days dengue viruses may circulate in the
peripheral blood (Gubler et al 1981a). Primary infection is thought to induce life long protective
immunity to the infecting serotype (Halstead SB 1974 ). Individuals suffering an infection are protected
from clinical illness with a different serotype within 2--3 months of the primary infection but with no
long-term cross-protective immunity. If other Ae. Aegypti mosquitoes bite the ill person during this
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febrile viremic stage, those mosquitoes may become infected and subsequently transmit the virus to other
uninfected persons after an extrinsic incubation period of 8 to 12 days (Gubler D.J 1976)
During a DEN 2 outbreak investigation in Tonga, despite the application of the most sensitive
virus isolation technique (mosquito inoculation) on blood specimens collected daily for as long as 6 days
and attempts to isolate virus by permitting uninfected mosquitoes to feed on patients, few virus strains
were isolated (Gubler et al 1978). It was speculated that the low virus isolation rate reflected a short
viremic period of low magnitude, corresponding with the abortive nature of the outbreak. This in turn
suggested that virus strains with low virulence were most likely the determining factor (Gubler et al
1978). A similar observation was documented in Indonesia with DEN 3 (Gubler et al 1981b) and in
Taiwan with an unidentified serotypes (Chen et al 1996).
1.6 Transmission of the dengue virus
Dengue virus circulating in the blood of viremic humans is ingested by female mosquitoes during
feeding. The virus then infects the mosquito mid-gut and subsequently spreads systemically over a period
of 8--12 days. After this extrinsic incubation period, the virus can be transmitted to other humans during
subsequent probing or feeding. The extrinsic incubation period is influenced in part by environmental
conditions, especially ambient temperature. Thereafter the mosquito remains infective for the rest of its
life. Ae. aegypti is one of the most efficient vectors for arboviruses because it is highly anthropophilic,
frequently bites several times before completing oogenesis, and thrives in close proximity to humans.
Vertical transmission (transovarial transmission) of dengue virus has been demonstrated in the laboratory
but rarely in the field. The significance of vertical transmission for maintenance of the virus is not well
understood. Sylvatic dengue strains in some parts of Africa and Asia may also lead to human infection,
causing mild illness. Several factors can influence the dynamics of virus transmission including
environmental and climate factors, host-pathogen interactions and population immunological factors.
Fig 1.6 Transmission of Dengue virus by Aedes aegypti

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1.6.1 Factors affecting the distribution of dengue vectors and transmission of Dengue viruses
Climate directly influences the biology of the vectors and thereby their abundance and
distribution; it is consequently an important determinant of vector-borne disease epidemics (dengue
guidelines for diagnosis, treatment, prevention and control, WHO 2009).
I) Rainfall
There is a strong positive correlation between dengue outbreak and rainfall pattern which
increases the number of breeding habitats of Aedes vectors (Li et al 1985). In semi arid areas i.e. India
Ae. aegypti is an urban vector and populations typically fluctuate with rainfall and water storage habits
(Kalra et al 1968). In other countries of South East Asia where the annual rainfall is greater than 200 cm,
Ae. aegypti populations are more stable and are established in urban, semi urban and rural areas. In
Indonesia, Myanmar and Thailand their densities are higher in semi urban areas than in urban areas due to
the traditional water storage practices (WHO SEARO 1999). There are some exceptions. In some
locations dengue outbreaks may occur and ceases before the arrival of the rainy season or occur in
relatively dry areas. Also even in locations with a rainy season, such as Singapore, sometimes a positive
correlation between rainfall and vector population density could not be obtained (WHO 1995b).
Further in parts of the tropics where there are two annual rainy seasons, a positive correlation was
observed in only one season (Aitken et al 1980). The impact of rainfall on adult vector density is not the
same for all vector species. Ae. aegypti which prefers indoor habitats is less affected by rainfall than Ae.
albopictus or other vectors that have outdoor larval habitats (Gubler et al 1997).
II) Temperature
Temperature is clearly a major factor controlling the seasonality of dengue outbreaks in sub
tropical or temperate regions. It influence on the vector distribution, blood feeding activity of the vector,
the extrinsic incubation period (EIP) and adult longevity. Ae. aegypti has been shown to transmit dengue
when the temperature was above 200C but not less than 160C. A study in Thailand has shown that
temperature is correlated with female abundance. In addition high temperature may increase frequent
blood feeding due to rapid reduction of energy reserves (Scott et al 2000b).
It is expected that the global warming may further facilitate the expanded distribution of dengue
mosquito vectors in temperate regions such as Northern parts of North America or Europe (Shope 1991).
This concern has become far more serious matter with the expanding distribution of Ae. albopictus
(Ward and Burgess 1993).
III) Altitude
Altitude is an important factor in limiting the distribution of Ae .aegypti. In India Ae.

aegypti ranges from sea level to 1000 meters above sea level. Lower elevations (less than 500 meters)
have moderate to heavy mosquito populations while mountainous area (greater than 500 meters) have low
populations (Kalra et al 1997).
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In countries of South East Asia, 1000 to 1500 meters appears to be the limit for Ae. agypti distribution
with some exceptions. In Colombia Ae. aegypti is known to survive indoors in locations at altitudes as
high as 2000m above WHO SEARO 1995).
IV) Transport of mosquito vector
The spread of viruses and vectors occurred via ships before the advent of air travel. Ae.
aegypti infestation of towns along the coast and rivers served by ships has been documented in South
America and Thailand (Soper 1967b, Pant et al 1973). Even though the ships were replaced by air planes
for transoceanic transportation of passengers and freight, they still play an important role in transporting
mosquito vectors today (Gubler D.J. & Kuno G. 1997).
Air travel has increased the incidence of dengue and the spread of viruses dramatically
during the past few decades. Survival of Ae. aegypti on flights originating from Puerto Rico to many
cities in the Caribbean basin was confirmed by the US Public Health Services as early as 1931 (Griffitts
1933). Survival of adult mosquitoes inside airplane cabins has been documented many times (Smith &
Carter 1984, Russell 1989).
Ground transportation also provides an efficient mechanism of dissemination of both

mosquito vectors and viruses (Soper 1967). In Trinidad it is strongly suspected that Ae aegypti gravid
females were unwittingly transported by passenger cars. The conclusion was based on continual positive
results of ovi traps in particular, in those set up near parking lots of an air port despite weekly removal of
positive paddles and immediate ultra low volume spraying of Malathion and larval control with temephos
at all positive sites during weekly surveillance ( Tikasingh 1991). The large number of automobiles used
for transportation in dengue endemic urban areas clearly favours rapid spread and accelerated contact
among humans vector and viruses
V) Threshold mosquito density for dengue transmission
The minimum mosquito density below which arbovirus disease transmission ceases has
been debated for many years without a clear resolution. House index for 1% has been selected as the
objective for yellow fever control in Senegal but it was not based on scientific studies (WHO 1972). In
Singapore where vector density has been held extremely low through a vigorous control programme
dengue haemorrhagic fever/dengue shock syndrome (DHF/DSS) outbreaks still occurred even when the
house index dropped to 1% (WHO 1992a).
It was also observed from many studies that larger number of mosquitoes per residence are not
always associated with epidemic transmission. In a localized school outbreak in Malaysia, only three Ae.
aegypti females were collected in one hostel where 20 students were infected (Smith 1956a).
In a study conducted during an outbreak in Taiwan, female density was 0.07 per house (Chen at
al 1994). Another important factor influencing transmission is the proportion of infective females.
Unfortunately there is paucity of data since virus has been isolated from pooled, rather than individual
mosquitoes.

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The minimum infection ratio obtained for Ae. aegypti and Ae. albopictus in Singapore in four sentinel
areas was, on average 0.51 and 0.59 per 1000 mosquitoes respectively (Chan et al 1971) while the
corresponding figures in another study in Thailand were 61 and 5 per 1000 respectively (Smith et al
1971).
VI) Flight range.
It was observed from mark release and recapture experiments that flight distance of Ae.
aegypti could range from 2.5km per day in an open environment (Wolfinsohn and Galun 1953) to a few
hundred meters (Reiter et al 1995) to less than 25m in an urban environment (Morlan and Hayes 1958).
Some studies strongly suggests the limited flight range of Ae. aegypti. During a small
outbreak of dengue in an isolated boarding school in Malaysia, none of the 600 students in the school
located only 50m from the site of the outbreak was affected (Smith 1956a).
In another study in an East African Village, the majority of marked Ae. aegypti remained
in the house where they were captured, and most females had visited one or two houses. Only 0.7 per cent
of them visited as many as five houses (Trpis and Haussermann 1986).
VII) Feeding behaviour
Female mosquitoes engage in multiple feeding before completing the gonotrophic cycle
(Gould et al 1970, Yasuno et al 1970). They are very nervous feeders, disrupting the feeding process at
the slightest movement, they return to the same or a different person to continue feeding moments later.
Because of this behaviour, Ae. aegypti females will often feed on several persons during
a single blood meal and if infective, may transmit dengue virus to multiple persons in a short time, even if
they only probe without taking blood meal . ( Gubler D.J., Rosen L. 976, Plat et al 1997, Putnam J.L. et al
1995 ).
This behaviour probably contributes to the explosive nature of dengue outbreaks and
may explain why relatively small numbers of infected females have been found in the residences in
endemic foci.
VIII) Extrinsic incubation period
It has been reported that extrinsic incubation period (EIP) ranges between 10 to 14 days
(Siler et al 1926, Sabin 1952). However it depends on ambient temperature, humidity, viremia level in the
human host and the virus strain.
Higher temperature results in a shorter EIP and on the other hand lower temperature
decreases transmission (Watts et al 1987).

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IX) Daily survival rate, longevity and gonotrophic cycle
A small change in daily survival rate has a considerable impact on transmission. For
example in a mathematical model of chikungunya transmitted by Ae. aegypti, a change in the daily
survival rate from 0.89 to 0.94 altered the course of the disease from a relatively brief, self limiting
epidemic to a stable endemic state (de Moor and Steffens 1970).
Most, if not all investigators working on mathematical models of arthropod borne

infections have accepted the assumption of Macdonald that the probability of mosquito survival is
constant throughout a mosquitos life. This idea was supported by a more recent publication (Clements
and Paterson 1981). According to Smith (1975) however , that is unlikely and needs further studies under
field conditions.
Longevity depends on many factors such as temperature, humidity and nutrition. Limited
data from field studies on female Ae. aegypti showed longevity ranging from 8.5 days (Sheppard et al
1969) to 42 days (Trpis and Hausermann 1986).
Under laboratory conditions however at 270C females that are given both a 10% sucrose
solution and blood meals survive on average, for 55.6 days with a maximum of 100 days (Briegel and
Kaiser 1973) . Females that are fed 5% sucrose solution and kept at ambient temperature in the tropics
transmit dengue 75 days after feeding on a viremic human (Siler et al 1926).
Ae. albopictus females are known to survive for up to 122 days in the laboratory (Gubler
1970), but there are no reliable field data where daily mortality varies from 7.9% to 15% depending on
the month (Gubler 1971). Temperature affects not only longevity but length of gonotrophic cycle which
was recognized to be a factor associated with seasonality of dengue in Bangkok (Pant et al 1973).

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Chapter 2
The vectors
2.1 Introduction
Globally, dengue and DHF is transmitted by female mosquitoes of the genus Aedes . In addition
to dengue and DHF, they are responsible for the transmission of
Yellow fever in Africa & tropical areas of America

Chikungunya
Other viral diseases.
Filariasis in some countries
Aedes mosquitoes occur around the world and there are over 950 species. They can cause a
serious biting nuisance to people and animals. There are 48 Aedes species in Sri Lanka but Aedes
(Stegomyia) aegypti (Linnaeus) and Aedes (Stegomyia) albopictus (Skuse) belonging to the sub genus
Stegomyia transmit dengue/DHF.
These vector mosquitoes are widely distributed around the world, mostly between latitudes 35
N and 35 S. These geographical limits correspond approximately to a winter isotherm of 10 C. Ae.
aegypti has been found as far north as 45 N, but such invasions have occurred during warmer months
and the mosquitoes have not been survived during the winters. Also, because of lower temperatures, Ae.
aegypti is relatively uncommon above 1000 metres.
A list of mosquito species associated with dengue virus transmission in different regions of the
globe are as follows.
i) Afrotropical Region (Subsaharan Africa) Selected reference
Aedes (Stegomyia) aegypti (Linnaeus) Trpis & Hausermann (1986)
Aedes (Stegomyia) albopictus (Skuse) Rodhain & Rosen (1997)
Aedes (Stegomyia) luteocephalus Rodhain & Rosen (1997)
Aedes (Stegomyia) opok Corbet & Van Someren Rodhain & Rosen (1997)
Aedes (Diceromyia) furcifer Edwards Rodhain & Rosen (1997)
Aedes (Diceromyia) taylori Edwards Rodhain & Rosen (1997)

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ii) South Pacific Islands and Australian Region Selected reference
Aedes (Stegomyia) aegypti (Linnaeus) Cleland et al (1916) Rodhain & Rosen (1997)
Aedes (Stegomyia) albopictus (Skuse) Belkin (1962a,b) Rodhain & Rosen (1997)
Aedes (Stegomyia) cooki Belkin Rodhain & Rosen (1997)
Aedes (Stegomyia) herbrideus Edwards Belkin (1962a,b) Rodhain & Rosen (1997)
Aedes (Stegomyia) hensili Farner Savage et al (1998) Rodhain & Rosen (1997)
Aedes (Stegomyia) polynesiensis Marks Rodhain & Rosen (1997)
Aedes (Stegomyia) rotumae Belkin Rodhain & Rosen (1997)
Aedes (Stegomyia) scutellaris (Walker) Rodhain & Rosen (1997)
Ochlerotatus (Finlaya) notoscriptus (Skuse) Rodhain & Rosen (1997)
iii) Oriental region including South East Asia
Aedes (Stegomyia) aegypti (Linnaeus) Hammon et al(1960), Huang(1961)
Rodhain & Rosen (1997)
Aedes (Stegomyia) albopictus (Skuse) Rudnick & Chan 1965, Smith et al (1971)
Huang (1979), Rodhain & Rosen (1997)
Ochlerotatus (Finlaya)niveus sub group Rudric (1986)
iv) Americas (including the Carribean Islands)
Aedes (Gymnometopa) mediovittatus (Coquillet ) Gubler et al (1985), freier and Rosen (1988)
Aedes (Stegomyia) aegypti (Linnaeus) Rodhain & Rosen (1997)
Aedes (Stegomyia) albopictus (Skuse) Rodhain & Rosen (1997)

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Aedes aegypti Aedes albopictus
Fig. 2.1 Adults of Ae. Aegypti and Ae. albopictus
2.2 Life cycle
The life cycle of an Aedes mosquito consist of 4 different stages (egg, lava, pupa and adult).
Fig 2.2 Life cycle of Aedes mosquito

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The immature stages (egg. Larva and pupa) are found in man made or natural, water-filled
container habitats, mostly in artificial containers closely associated with human dwellings, both indoors
and outdoors.
2.2.1 Eggs
Egg shell has a mosaic pattern

Eggs are deposited singly on damp surfaces on or just above the water line in containers.
Most females lay eggs in several oviposition sites during a single gonotrophic cycle.
Embryonic development is usually completed in 48 hours in a warm and humid environment.
The eggs can withstand long periods of desiccation (more than a year).
Eggs hatch once the containers are flooded, but not all eggs hatch at the same time.
The capacity of eggs to withstand desiccation, facilitates the survival of the species during adverse
climatic conditions .During such long periods the containers containing the eggs could be tranported
from place to place, even from country to country (Ae albopictus was introduced to America by this
method)
Fig 2.3 Eggs of Aedes
2.2.2. Larva
The larvae pass through four developmental stages, 1st Instar, 2nd Instar, 3rd Instar, 4th Instar.
The duration of larval development depends on
temperature,
availability of food, and
larval density in the receptacle.
Under optimal conditions, the time taken from hatching of egg to adult emergence can be as short as
seven days, including two days in the pupal stage. At low temperatures, however, it may take several
weeks for adults to emerge.

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Fig. 2.4 Aedes Larvae Fig. 2.5 Last abdominal segment of larva
2.2.3. Pupa:
The 4th stage larva become pupa and the pupa do not feed but respire through the respiratory trumpets.
The adult mosquito emerges from the pupa within 48 hrs.
Fig 2.6 Pupa
2.2.4 Adult
Many species of the Aedes adults have conspicuous patterns on the thorax formed by black white or
silvery scales. The legs often have black and white rings. Abdomen is pointed. Adults of Ae. aegypti and
Ae. albopictus can be easily identified with the silvery ornamentation on the thorax.
Soon after emergence from pupa, the adult mosquitoes mate and the inseminated female may take a
blood meal within 24-36 hours. Blood is the source of protein essential for the maturation of eggs. Most
females lay eggs in several oviposition sites during a single gonotrophic cycle.

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Fig 2.7 Aedes aegypti Aedes albopictus
2.3 Main characters of identification of the genus Aedes
2.3.1 adults
1. . Prespiracular setae (PsS) absent

2. Postspiracular setae (PS) nearly always present
3. Mesopostnotal setae (MpnS) usually absent
4. Base of hindcoxa (C-III) usually below base of mesomeron (Msm)
5. Wing remigial setae (ReS) absent
6. Abdomen apex usually, but not always, more or less pointed in females
Fig 2.8 Main characters of identification of the genus Aedes Adults
1. Prespiracular setae (PsS) absent 2. Postspiracular setae (PS) nearly always present

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3. Mesopostnotal setae (MpnS) usually 4. Base of hind coxa (C-III) usually below
absent base of mesomeron (Msm)
5. Wing remigial setae (ReS) absent 6. Abdomen apex usually but not always
more or less pointed in females

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2.3.2 Main characters of identification of the genus Aedes Larvae
Fig 2.9 Aedes Larva
1. Metathoracic pleural group of setae (9-12-T) well developed

2. Pecten (Pt) always present (may be reduced to one or two spines)
3. Single pair of seta 1-S, inserted near or beyond middle of siphon (S )
Fig 2.10 Main characters of identification of the genus Aedes (Larva)
1.Metathoracic pleural group of setae 2. Pecten (Pt) always present (may be

(9-12-T) well developed reduced to one or two spines)

20
3. Single pair of seta 1-S, inserted near or beyond middle of siphon (S)
2.4. Breeding sites
Breeding sites are man-made or natural water receptacles. These include a multitude of receptacles and
roof gutters found in and around both urban and rural environments (households, construction sites and
factories etc) . A comprehensive list of breeding sites that are commonly found are
Tyres
water storage tanks / barrels, containers,
Machinery parts
Discarded receptacles such as tins, plastic ware, glass ware, coconut shells etc.
Flower pots & flower pot trays
Bird baths
Earth pipes
Ant traps
Flower vases,
Leaf axils
Bamboo stumps
Tree holes
Non used boats
Blocked roof gutters

21
Fig 2.11 Common Breeding sites of dengue vector mosquitoes
i) Ornamental / recreational (plant pots and dishes, rooting plants in water, aquatic
plants,

22
ii. Utensils / tools / toys (trash cans, pails or buckets, tarps coolers, painting trays)
iii) plastic pools

23
iv) Water-storage (wells, tanks, cisterns, barrels, jars, buckets)

24
v.) Large discarded containers (damaged appliances, tires, etc)
v) Leaking water meters vii) Animal drinking pans

25
Viii) Fallen leaves

26
ix) Leaf axils
x) Tree holes

27
xi) Bamboo stumps xii) Blocked roof gutters
The major breeding sites of Ae. aegypti and Ae albopictus varies from area to area, time to time and
season to season (wet & dry). In Sri Lanka, discarded receptacles are the predominant breeding sites in
many districts of the country but in Hambantota, Kandy and Matale districts, majority of the breeding
sites comprised of water storage tanks and containers.
plant axils tree holes

5% 2% cement tanks
tyres 3%
10%
plastic
41%
water storage barrels
12%
other discarded
receptacles
3%
clay pot coconut shell

bird bath 8% 10%
tins
2%
4%
Blocked roof gutters not included

Fig 2.12 Percentage prevalence of breeding sites in the Western province Sri Lanka 2010

28
2.5 Bionomics of the adults
2.5.1 Feeding behaviour of Aedes aegypti and Aedes albopictus
They are highly anthropophilic

Day time feeders
Blood feeding during day time may be interrupted. They move from person to person and feed on
more than one person. This multiple feeding behaviour greatly increases the epidemic transmission
efficiency. Thus, it is not uncommon to see several members of the same household with an onset of
illness occurring within 24 hours, suggesting that they were infected by the same infective mosquito. Ae.
aegypti generally does not bite at night, but it will feed at night in lighted rooms (Lumsden 1957).
Ae. aegypti and Ae albopictus have two periods of biting activity , one in the morning for several
hours after day break and the other in the afternoon for several hours before dark (Nelson et al 1978,
Lumsden 1957, Sheppard 1969). The actual peak of biting activity may vary with location and season.
Studies carried out in Tangalle district and western province has shown that variation in biting habits of
Ae. aegypti and Ae. albopictus (fig 2.13)
Fig 2.13 (i), (ii), (iii), (iv) Biting patterns of Aedes aegypti and Aedes albopictus in different locations
(i)
Biting Behaviour of Aedes aegypti in Tangalle
1.600
landed/hr/bait
1.400
/ hbt
1.200
1.000
landing mosquito
0.800
of mosquitoes
0.600
0.400
No.of
0.200
No.
0.000
,5-6 ,6-7 ,7-8 ,8-9 ,9-10 ,10-11 ,11-12 ,12-13 ,13-14 ,14-15 ,15-16 ,16-17 ,17-18 ,18-19 ,19-20
Hour
Indoor Outdoor

29
ii)
Biting Behaviour of Aedes albopictus in Tangalle
1.600
1.400
No. of mosquitoes landed/hr/bait
1.200
1.000
0.800
No.od landing mosquitos/hr/bait
0.600
0.400
0.200
0.000
,5-6 ,6-7 ,7-8 ,8-9 ,9-10 ,10-11 ,11-12 ,12-13 ,13-14 ,14-15 ,15-16 ,16-17 ,17-18 ,18-19 ,19-20
Hour
Indoor Outdoor
iii) Human Landing collection in a open space (bus depot) in the Western Province
35
30
landed/hr/bait
mos. landed/person
25
20
15
No.ofofmosquitoes
10
5
0
No.
Ae.aegypti Ae.albopictus

30
iv) Human Landing collection in a shady area (in a garden with lot of trees) in the Western
Province
9
landed/ person
8
No. mos.landed/hr/bait
7
6
5
4
3
No. of mosquitoes
2
1
0
Ae.aegypti Ae.albopictus
2.5.2 Resting behaviour
Aedes aegypti and Aedes albopictus rest both indoors and outdoors.
Indoors - in dark, humid, secluded places inside houses or buildings, including bedrooms, closets,
bathrooms and kitchens. The preferred indoor resting surfaces are the undersides of
furniture, hanging objects such as clothes and curtains, and on walls.
Outdoors - vegetation or other protected sites.
2.5.3 Flight range and dispersal
The dispersal of adult female is influenced by a number of factors including availability of ovi-position
sites and blood meals, but appears to be often limited to within 100 meters of the site of emergence.
However recent studies in Puerto Rico indicate that they may disperse more than 400 meters primarily in
search of ovi-position sites. Passive transportation can occur via eggs and larvae in containers.
Studies suggest that most female Ae. aegypti may spend their lifetime in or around the houses where they
emerge as adults. This means that people, rather than mosquitoes, rapidly move the virus within and
between communities. Dengue outbreaks have also been attributed to Ae. albopictus, Ae. polynesiensis
and several species of the Ae. scutellaris complex.

31
Each of these species has a particular ecology, behaviour and geographical distribution. In recent decades
Ae. albopictus has spread from Asia to Africa, the Americas and Europe, notably aided by the
international trade in used tyres in which eggs are deposited when they contain rainwater. (dengue
guidelines for diagnosis, treatment, prevention and control, WHO 2009).
2.5.4 Longevity
They have an average adult survival of only eight days. During the rainy season, when survival is longer,
the risk of virus transmission is greater. More research is required on the natural survival under various
environmental conditions.
2.6 Human factors influencing the distribution of Dengue
Dengue and dengue haemorrhagic fever emerged as a major public health problem during the 20th
century and the reasons are complex and not fully understood. However several important factors are
identified (Gubler & Trent 1994).
1) Introduction of dengue virus to susceptible population
The re-infestation of the American by tropics, Ae. aegypti provided a pool of susceptible
individuals living in urban areas that approximates 300 million. The numerous epidemics of dengue that
subsequently occurred provided increased opportunity for the viruses to move between countries , both
within and out of the region.
2) Non existence of effective vector control measures
Effective mosquito control is virtually non existent in most dengue endemic countries of the
world. Emphasis for the past 20 years has been placed on Ultra Low Volume (ULV) space sprays of
insecticides for adult mosquito control (Gubler 1989). This has been shown to be an ineffective in
controlling Ae. aegypti (Gubler 1989, Newton and Reiter 1992).
3) Demographical Changes
Major global demographic changes have occurred, the most important of which have been
concurrent uncontrolled population growth and unplanned urbanization. These changes have resulted in
sub standard housing and inadequate water and waste management systems for millions of people in
urban centres of the tropics.
4) Use of non bio degradable containers for food storage
Most consumer goods are packed in non biodegradable plastic or cellophane which is discarded
into the environment where it provides ideal larval habitats for the vector mosquito.

32
5) Automobile Tyres
Used automobile tyres which have increased dramatically in numbers in the past 20 years and
which are very difficult to dispose of, from the environment makes another important breeding site for
dengue mosquitoes (Gubler & Kuno 1997).
6) Water storage
Cultural difference in the practice of using or storing water in living premises also influence
vector density.
7) Increased travel of humans
Increased travel of humans by air plane provides the ideal mechanism for transporting dengue
viruses between population centres of the tropics, resulting in a constant exchange of dengue viruses.
For example from 1983 1994 , the number of international departures from US airports doubled from 20
to 40 million and over 50% of these departures each year are to tropical areas (Gubler 1996).
8) Passive transportation of mosquitoes by vehicles
Passive transportation of adult mosquitoes by vehicles, aero planes and ships is another factor
influencing the distribution of dengue. Survival of Ae. aegypti on flights originating from Puerto Rico to
many cities in the Caribbean basin was confirmed by the US Public Health Services as early as 1931
(Griffitts 1933). Survival of adult mosquitoes inside airplane cabins has been documented many times
(Smith & Carter 1984, Russell 1989).
All these factors have contributed to the expanded geographic distribution and increased
densities of the dengue mosquito vectors.

33
2.7 Household transmission
Once an infective mosquito enters a house or a member of a household becomes infected, the probability
of multiple infection in the household increases and may eventually results in clusters of dengue
infections (Gubler & Kuno 1997).
2.7.1 Human movement and exposure to virus
i) In endemic areas
Disease spread is a function of the probability of contact between human, virus and mosquito.
Therefore transmission is facilitated in any Ae. aegypti infested location where people congregate
resulting in either transmission from infective mosquito to human or from viremic human to uninfected
mosquito. Hospitals without protective measure against mosquitoes were suspected as foci of
transmission (Siler et al 1926). Halstead (1984) pointed out the importance of the movement of infected
children as a mechanism of dengue spread. In an isolated village in Thailand a buddhist temple that had a
school for boys was a primary focus of transmission (Silarug et al 1990). The radial spread from the
initial focus along busy routes of human traffic has been documented many times in Asia ( Dizon et al
1966, Kalra et al 1966). thus wherever people congregate , including gatherings of neighbours, friends or
relatives the probability of contact among virus and female mosquito accelerates. The influx of a large
number of dengue nave individuals to dengue endemic areas often results in outbreaks as typically
occurs when a large number of foreigners settle or soldiers are deployed in dengue endemic locations.
Migration of susceptible labourers from the North East to the South in Thailand is strongly suspected to
be the cause of increased dengue incidence in the Southern provinces (Sucharit 1993).
ii) In vector infested but dengue free areas

Historically humans have been responsible for the dissemination of both vector and virus. Once
infested with vectors, the area is at risk of dengue outbreaks if the virus is introduced. Early dengue
spread in Australia was presumably caused by train passengers carrying the virus (Wolstenholme 1993).
Return of a large number of sick seamen from South East Asia and the Pacific was responsible for dengue
outbreaks in port cities in Japan (Hotta 1953).
2.8 Vector competence
Vector competence (VC) is defined as the intrinsic permissiveness of a vector to infection,

replication, and transmission of a virus (Hardy 1988).
These include
i) a mid gut infection barrier (MIB),
ii) a mid gut escape barrier (MEB) and
iii)a salivary gland barrier (Woodring 1996).

34
In potential vectors with MIB, virus cannot infect and/or replicate in the mosquito mid gut cells.
This may be due to lack of cell surface receptors for the virus or to mid gut cells being non permissive for
infection with the virus.
Potential vectors with MEB may allow virus replication in the mid gut , even to high titers but
virus is then unable to exit the mid gut to cause disseminated infection. Barriers to infection can vary
widely among Ae. aegypti populations causing vector competence for dengue viruses to be variable as
well (Kristine E. Bennett et al 2002).
In a quantitative genetic study of the ability of Ae. aegypti to propagate DEN 2 virus in the mid
gut and in a disseminated infection in the head, it was observed that once infection of the mid gut occurs,
the amount of virus propagated in the mid gut epithelial cells was the same between sub species.
Similarly once the infection had escaped the mid gut, the amount of virus propagated in the head was
also the same in sub species. Further more the amount of virus in the midgut did not influence if or how
much virus would escape from the mid gut. It is the barriers to infection and dissemination independent
of virus titer, that determines vector competence for Den 2 virus ( Bosio C.F. et al 1998). This is
consistent with the findings of Miller and Mitchell (1991) in which yellow fever virus susceptible and
resistant lines of Ae. aegypti were similar in virus titer for the first five days after oral infection, after
which the susceptible line continued to increase to high titer, while the resistant line did not.
From this study they concluded that the replication occurs in the mid gut of both lines at
equal levels. The fact that viral titers in infected tissues did not differ between sub species suggests that
genes that influence virus titer in the mid gut or other body tissues ultimately have a minimal impact on
the overall vector competence of Ae. aegypti for DEN 2 virus. Instead it is the genes that control the MIB
and the MEB that exert the greatest effect on vector competence.
Ae. aegypti populations exhibit considerable genetic variability in vector competence for
flavivirus (Aitken et al 1977, Tabachnick 1985, Tabachnick 1979, Rosen L. et al 1985). The vector
competence for flaviviruses in Ae. aegypti is thought to be controlled by at least two genes or sets of
genes, one controlling the MIB and the other controlling the MEB (Miler et al 1991, Bosio et al 1988,
Bosio et al 2000).
The primary vector of dengue viruses is the mosquito Ae. aegypti. Sub species and strains of Ae.
aegypti have been shown to vary phenotypically in their susceptibility to DEN viruses (Gubler D.J. et al
1979). Ae. aegypti has a broad geographic distribution and many studies have shown that it is a more
competent vector of flaviviruses than Ae. aegypti fermosus, which is distributed only in sylvan and rural
areas of West Africa (Gubler D.J. et al 1979, Tabanick W.J. et al 1985). Similar phenotypic variation
has been seen for DEN viruses in another important vector Ae. albopictus (Gubler D.J. et al 1976).
It was observed from several studies that oral receptivity of Ae. aegypti mosquitoes from
different parts of the world where dengue is endemic ( South Vietnam) or epidemic (French Polynesia,
French Guinea), was very high but the oral receptivity of Ae. albopictus was very low (Marie Vazeille
2003, Tran Khanh et al 1999, Vazeille Falcoz M. et al 1999, Fouque F et al 2001, Paupy C. et al 2001).

35
However these results contradict the generally accepted belief that Ae. aegypti is much less
receptive to oral infection with dengue viruses of all four types than are most other Aedes capable of
transmitting such viruses (Rodhain F. et al 1997, Rosen L et al 1985).
Environmental conditions also affect vector competence . Larval crowding and nutrition as well as age of
the adult mosquito significantly affect vector competence for arboviruses (Baqar S et al 1980,
Grimstead P.R. 1984).
Other studies have demonstrated the importance of temperature in several vector systems
including the vector competence of Ae. aegypti for DEN virus (Watts D.M. et al 1987).

36
Chapter 3
Dengue Vector Surveillance
3.1 Introduction
Surveillance is defined as the analysis and interpretation of systematically collected data for facilitating
timely and appropriate decision on vector control.
Surveillance programmes are essential for planning, operational, monitoring and evaluation of vector
control activities.
The selection and use of method requires a clear understanding of the surveillance objectives, the
availability of skills and resources, and in some instances the level of infestation. There are several pre-
requisites to the development of vector surveillance activities. The most important is an understanding of
the biology of the vectors, including vector competency to transmit disease, breeding sites, and its spatial
and temporal distribution.
The main purpose of surveys for dengue vector surveillance is to obtain information that can be used to
control population densities of Aedes vector.
Those information will be helpful to :
1. determine the key breeding sites in the domestic and peri-domestic environments.
2. determine the species composition in the area
3. determine seasonal population fluctuations
4. determine the bionomics of the vector (feeding, biting, resting habits)
5. forecast the possible dengue outbreaks/epidemics.
6. determine the appropriate vector control interventions
7. determine the optimum time for the application of vector control measures
8. determine the impact of vector control activities
9. determine geo-spatial and temporal distribution of dengue vectors.
10. status of the susceptibility to insecticides
In areas where the vector is no longer present, entomological surveillance is critical in order to detect new
introductions rapidly before they become widespread and difficult to eliminate.
3.2 Types of surveys
i) Spot checks
ii) Sentinel site surveys

37
i) Spot checks
Spot check is carried out
a) When there is an increase in the reporting of fever /suspected dengue cases, in a particular
locality is identified
b) Where there is a continuous reporting of dengue cases , despite of all the vector control
efforts
c) When environment changes occur favouring vector breeding (eg. flooding, development
projects etc)
d) When there is a need to evaluate the impact of control measures (eg: cleaning progammes,
fogging etc)
e) To identify the new establishment of Aedes aegypti or Aedes albopictus in areas where there
were no reports of the vector previously.
iii) Sentinel site surveys (Regular or Trend observation)
a) Observations are carried out regularly, weekly, fortnightly, or monthly depending on the
requirement and availability of human resources.
b) Area is selected on the past history of dengue cases or based on the high vector density.
c) In potential areas with environmental changes
d) To observe changes in vector density, biology and behavior
e) To monitor susceptibility/resistance levels of vectors to insecticides
3.3 The essential components in a dengue vector survey:
1. Planning survey
2. Sampling of mosquitoes
3. Identification of mosquitoes
4. Determination of vector indices
5. Determination of vector densities
6. Interpretation of results
3.3.1 Sampling of mosquitoes
Knowledgeable, skilled and interested personnel are of paramount importance in a good sampling
programme. Training of the field staff is a key element in obtaining this goal. Field personnel need to
be able to identify the difference between mosquito larvae, pupae and mosquito-like diptera.

38
They should also be familiar with the major nuisance and vector mosquitoes encountered in the area,
their seasonal occurrence and typical breeding habitats.
3.3.2 Sampling strategies
Only in exceptional conditions, larval surveys of every house is (i.e. census) warranted. Such
situations arise when the objective is vector eradication or bringing down the larval infestation levels to
very low levels (HI = <1.0%). At this point it is necessary to locate and control every infested and
potentially infested container, or to verify that eradication has indeed been achieved, or to ensure that re-
infestation has not occurred.
In other situations, the number of houses to be inspected should be based on
i) considerations of available resources,

ii) the desired level of precision of the results, and
iii) the total number of houses in the locality
This is contrary to the routine procedures employed in many vector control programmes in which
eradication campaign methodologies are persisted and entomological data are collected from every house
immediately prior to insecticide treatment, usually by the person who administered the treatment. Such
practices, if used solely for measuring infestation levels, are wasteful and are likely to result in poor
quality reporting due to conflicts of worker interest and the tedious nature of the work.
Whenever possible, it is recommended that a different team or individual should conduct the
entomological evaluation, or that the two tasks should be performed separately. The sample size for
routine surveys can be calculated by statistical methods based on the expected level of infestation and the
desired level of confidence in the results.
Several sampling procedures that eliminate or minimize bias can be applied equally well, to the
selection of houses for larval, adult, ovi trap, or knowledge-attitude-practice (KAP) surveys.
These are as follows:
Systematic sampling of every nth house throughout a community or along a linear transect through
the community is the widely accepted method. For example, if sample of 25% of houses is to be
examined, every 4th house would be inspected.
This is a practical option for rapid assessment of infestation levels, especially in areas where there is no
house-numbering system. All areas of the locality are well represented.
Simple random sampling means that houses to be selected are obtained from a list of random numbers
(either from tables of random numbers in a statistical textbook or from a calculator or a computer-
generated list). This is a more laborious process since detailed house maps or lists of street addresses are a
prerequisite for selecting the houses. Many statistical tests require random sampling. Unfortunately,

39
although every house has an equal chance of being selected, some areas of the locality are usually under-
represented and others are over-represented.
Stratified random sampling minimizes the problem of under-representation and over-representation by

subdividing the localities into sectors or strata. These are usually based on identified risk factors such
as areas with houses without a piped water supply, areas not served by sanitation services, and densely
populated areas. A simple random sample is taken from each stratum, with the number of houses
inspected being in proportion to the number of houses in each stratum.
Cluster sampling may be conducted in large cities or geographical areas where it may be difficult or
impossible to use random or systematic sampling because of limitations of time, money and personnel, or
because of other logistical constraints. In these circumstances, the sample may be selected in two stages in
order to minimize the resources needed for the survey. The first stage is obtained by simple or stratified
random sampling of population groups or clusters (e.g. city blocks, villages, or administrative districts).
Having identified these clusters, simple or stratified random sampling procedures are again applied to
identify the specific houses within each cluster for inclusion in the survey.
Depending primarily on the objective of the survey, there are three basic methods;
* sampling of larvae /pupae
* sampling of adult
* sampling of ovi positing population
3.4 Sampling of larvae
For reasons of practicality and reproducibility, the most common survey methodologies employ larval
(active immatures, including pupae) sampling procedures rather than egg or adult collections. The basic
sampling unit is the house or premise, which is systematically searched for water-holding containers.
Containers are examined for the presence of mosquito larvae, pupae, and larval and pupal skins.
Laboratory examination is necessary to confirm the species
3.4.1 Larval sampling procedure
3.4.1.1 The basic tools required for larval surveys
a standard, white 400 ml-capacity dipper / with extendable handle.

a small pipette
vials, 6 oz plastic bags or some other form of container for collecting larvae
Torch
labels for the collections; and a pencil.
70% Ethyl alcohol or 10% formaldehyde
40
Ladder
Mirror with extendable handle
Other, more specialized tools may be necessary for sampling larval habitats inaccessible to a
dipper.
3.4.1.2- Method of survey
Individual households/premises are the appropriate spatial unit for these surveys. All
houses/premises within at least a radius of 200 m of the dengue case /focal point must be totally
examined for Aedes breeding and the quality of the surveys should be ensured. Ideally such
surveys must be carried out within 24 hours of notification of the first case from an outbreak
locality.
When the human population density is high in the selected area a representative sample of
minimum of 100 houses/premises could be examined.
Several sampling procedures that eliminate, bias could be used for the selection of houses for
larval surveys.
During larval surveys all receptacles should be visually examined for evidence of vector larvae,
pupae or eggs.
At each premises the name of occupant or establishment, address, types of containers with water
collections, number of containers with larvae should be documented.
The larval and pupal density or number of larvae and pupae in each breeding site could be noted
down.
When dip net is used for sampling in water storage tanks etc, five samples i.e., two samples from
the surface and three samples from the middle and the bottom should be taken.
A sample of larvae, at least 10 larvae per breeding site, ( if the no. of larvae is less than 10, all
larvae should be collected) should be taken from each positive breeding site for identification
purposes.
If there are a lot of breeding sites of same type in the same premise, maximum 10 randomly
selected breeding sites should be examined.
A drop of formaldehyde or ethanol should be added to the vials containing the sample to preserve
the specimens.
All collected larvae and pupae should be identified into species.
The breteau index, container index and premise index should be calculated for each survey
carried out.

41
The larval stages of Aedes mosquitoes occur in various habitats. Various techniques can be used to collect
them from different types of habitats.
These include dipping, pipetting, siphoning, etc.
i) Dipping
The dipper is the most commonly used tool for collecting larvae occurring in large and small water
bodies. Dipping usually catch larvae at the water surface and at different levels of the water column.
Fig. 3.1 Dipping using a ladle
Method
Skim a dipper at an angle quickly through the water and remove before it overflows or gently lower
at one point and allow water to flow into it.
Lift the dipper and hold it steady to allow the larvae to come up.
Pick them up by pipettes and transfer them into bottles or vials.

42
ii) Pipetting
Pipetting collect the larvae from small water collections where dipper cannot be used.
Fig. 3.2 pipetting

43
iii) Siphoning used for sampling from tree holes, leaf axils
Fig 3.4 siphoning
iv) Netting/funnel traps used for sampling from wells, cement tanks (specially from large volume
of water)
Nets are mounted on a frame and attached to a handle. They are used to sample larvae in
large habitats, e.g. burrow pits, wells etc.
Calibration of the device, using known numbers of Ae. aegypti larvae, enables the size of
the larval population to be estimated .
In some locations the device has focused attention on the importance of subterranean
habitats and harbourages during winter or in dry conditions . The funnel trap captures a
lower proportion of pupae because they are less active than larvae.
Quantification of the funnel trap allows results to be compared with larval counts in other
containers and allows estimates to be made of the relative importance of the various
types of containers.
However, there is no way to relate funnel trap captures to the risk of transmission because
there is no direct relationship between larval densities and density dependent larval
survival.

44
Fig. 3.5 Netting / funnel trap
3.4.2 Calculation of Indices
Following three indices would be calculated to determine the larval density levels of the particular
locality:
1. House/Premise Index (HI)
Percentage of houses or premises infested with larvae/ or pupae
HI = Infested houses
X 100
Houses inspected
2. Container Index (CI)
Percentage of water holding containers infested with Aedes larvae / or pupae
CI = Containers positive
X 100
Containers inspected

45
3. Breteau Index (BI)
Number of positive containers per 100 houses inspected
BI = No. of positive containers

X 100
Houses examined
Eg:
No. houses No. houses positive for No. of containers No. containers positive for
examined examined
A B A& B (mixed A B A& B (mixed
breeding) breeding)
100 05 03 04 50 08 06 02
No. houses infested with A

House Index for Ae. aegypti (A) = X 100
No. Houses
100 examined
= (05 + 04) x 100
100
House Index for Ae. albopictus (B) = No. houses infested with B
X 100
No. Houses examined
= (03 + 04) x 100
100

46
Container Index for Ae. aegypti (A) No. containers infested with A
X 100
No. containers examined
= (08 + 02) x 100
50
Container Index for Ae. albopictus (B) No. containers infested with B
X 100
No. containers examined
= (06 + 02) x 100
50
Breteau index for Ae. aegypti (A) No. of positive containers with A
X 100
No. Houses examined
= (08 + 02) x 100
100
No. of positive containers with B

Breteau index for Ae. albopictus (B) X 100
No. Houses examined
= (06 + 02) x 100
100

47
4. Common breteau index:
No. of houses examined and total no. of containers positive for Ae. aegypti, Ae. albopictus and no. of
containers with mixed breeding should be considered when calculating the common breteau index.
Eg:
Common breteau index = No. of containers positive for ( A + B + AB )

X 100
No. of houses examined
House index has been used most widely for measuring population levels, but it does not take into
account the number of positive containers or the productivity of those containers.
Container index provides information only on the proportion of water-holding containers that are
positive and most important container type.
Breteau index establishes a relationship between positive containers and houses, and is
considered to be the most informative, but again there is no accommodation of container
productivity.
While gathering the basic information for calculating the Breteau index, it is possible (and highly
desirable) to obtain a profile of the larval habitat characteristics by recording the relative
abundance of the various container types either as potential or actual sites of mosquito production
(e.g. the number of positive drums per 100 houses, the number of positive tyres per 100 houses).
These data are particularly relevant for focusing larval control efforts on the management or
elimination of the most common habitats and for the orientation of educational messages for
community-based initiatives.
A problem here is that the most common container (e.g. the plastic ware) is often not the most
productive. It should be noted that larval indices are a poor indication of adult production. For
instance, the rate of emergence of adult mosquitoes from rainwater drums is likely to differ
markedly from the rate from discarded cans or house plants, yet the larval survey registers them
only as positive or negative. The implication is that for localities with similar larval indices but
different container profiles, adult abundance and hence transmission potentials may be quite
different.

48
3.5 Pupal /demographic Surveys
The pupal/demographic survey is a method of identifying the most epidemiologically important

types of containers and may therefore be considered an operational research tool .
Unlike the traditional Stegomyia (Aedes) indices described above, pupal/demographic surveys
measure the total number of pupae in different classes of containers in a given community.
Such surveys are far more labour intensive than the above-mentioned larval surveys and are not
envisaged for the routine monitoring of Ae. aegypti populations.
The collection of demographic data enables the calculation of the ratio between the numbers of
pupae (a proxy for adult mosquitoes) and persons in the community.
There is growing evidence that, together with other epidemiological parameters such as dengue
serotype- specific sero-conversion rates and temperature, it is possible to determine how much
vector control is needed in a specific location to inhibit virus transmission.
This remains an important area for research with potential for public health application. Similar
methods have been used to measure total populations of larvae.
3.5.1 Pupal survey
The rate of contribution of newly emerged adults to the adult mosquito population from different
container types can vary widely. The estimation of relative adult production based on pupal counts
(counting all pupae found in each container) will help to identify the most productive containers which
will be important for the control programme. The corresponding index is the pupal index
3.5.2 Pupal counting has practical difficulties
It is labour intensive when obtaining pupal counts especially from large containers
Therefore it is difficult to be used in every survey,
It may be used for special studies or can be used once in each locality during the wet season and
once during the dry season, to determine the most productive container types.
Pupal Index (PI) = Number of pupae per 100 premises
PI = Total No. of pupae

X 100
No. of houses inspected

49
3.6 Adult Surveys
Adult vector sampling can provide valuable data for studies of seasonal population trends or evaluation of
adulticiding measures. However, results are less reproducible than those obtained from sampling of
immature stages. The methods for collection of adult mosquitoes also tend to be labour - intensive and
depend heavily on the collectors proficiency and skill.
Methods used are
Landing collections
resting collections using aspirators / back pack aspirators
Collections using resting boxes
use of sticky tapes
3.6.1 Landing collections
Landing collections on humans are a sensitive means of detecting low-level infestations.

Used for studying the biting times and places of host attraction.
Labour-intensive and expensive.
As there is no prophylaxis for dengue or other viruses transmitted by Aedes mosquitoes, has
safety and ethical issues in areas endemic for disease.
Both male and female Ae. aegypti are attracted to humans. Because adult mosquitoes, especially
males, have low dispersal rates, their presence can be a reliable indicator of proximity to hidden
larval habitats.
The rates of capture, typically using hand nets or aspirators as mosquitoes approach or land on the
collector, are usually expressed in terms of landing/biting counts per man hour.
Two types
1. Human landing catch

2. Indirect catch using double trap method
3.6.1.1. Human landing collections
Female mosquitoes are attracted to human to obtain blood meals. The number of vectors biting humans is
a major determinant of a disease transmission.

50
In dengue control operations it is also necessary to determine whether there are changes in
The aedes species biting humans

The number of vectors biting human
The number of vectors biting human indoors compared with the number biting in outdoors
(i) Essential equipments
a sucking tube
A test tube
Paper cups with net covers
Cotton wool
Towel
An insulated picnic box
A pencil
A note book
(ii) Rules
Do not smoke while collection.

Change the people being used as human bait hourly so as to minimize possible differences in their
attractiveness to mosquitoes.
Do not use any oil or ointment that might act as a repellant.
(iii) Method
Select a quiet place either inside or outdoors.

Adjust your clothes so that your legs are exposed as far as your knee, then sit quietly.
When you see mosquito landing on your leg, collect it with your sucking tube or test tube. Use
one cup for each hour of collection.

51
Fig. 3.6 Human landing catch
(iv) Labeling recording and preserving hourly collections
The paper cups containing the hourly collection must be very clearly labeled
Location
Date
Type of bait
Site of collection (indoor/outdoor)
Hour of collection
The collectors name
(v) Transportation to the laboratory
Collections should be transported to the laboratory in picnic boxes.
(vi) Calculation of indices
Man Hour Density = No. of mosquito species landed
No. of hours taken

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3.6.1.2 Human baited net traps
(i) Collection site
Two trap nets will be setup in one in a sleeping room and the second one outdoors at a site where people
regularly sit during the evening or sleep.
(ii) Collection using human baited trap nets
Set up human baited net trap as shown in Fig 3.7.

Set up a folding bed.
Put up the inner net around the bed to protect the person acting as a bait.
Erect the outer net.
Stretch the bottom of the outer net tightly and tie it to pegs in the ground ; leave 15-20 cm
between the ground and the lower edge of the net.
At sun rise get into the trap and lie down on the bed.
Set the alarm clock to ring after one hour. The collection period should not exceed 10 minutes
When the alarm ring collect all the mosquitoes in the trap net.
Transfer the mosquitoes to a container and label with the date, time, location and name. Use one
container for each collection per hour.
Repeat the procedure throughout the collecting period.
Fig. 3.7 Indirect catch using double trap method

53
3.6.2 Resting collections
During periods of inactivity, adult mosquitoes typically rests indoors, especially in bedrooms,
and mostly in dark places such as clothes closets and other hidden sites.
Resting collections involve the systematic searching of these sites with the aid of a flashlight and
the capture of adults using mouth- or battery-powered aspirators an hand-held net.
Resting collections could be done by using sucking tubes or backpack aspirators.
Densities are recorded as the number of adult mosquitoes per house (females, males, or both) or
the number of adults per man-hour of effort. Where infestation levels are low, the percentage of
houses positive for adults is sometimes used.
Resting collections provide information about the biology and behaviour of the vectors and are
useful in monitoring the efficacy of vector control programmes.
It also useful in the evaluating the efficacy of ultra-low volume space spray programmes directed
against vector mosquito species.
i) Use of a sucking tube
With the mouthpiece in your mouth, hold the sucking tube with its opening 1-2 cm away from
the mosquito.
Move the end of the sucking tube closer to the mosquito and suck gently but quickly so as to
draw the mosquito into the tube.
Place your finger over the tube to prevent the mosquito from escaping.
Transfer the mosquito to a paper cup by gently blowing into the mouthpiece (Those paper
cups containing live mosquitoes must have a wad of cotton wool moistened with
10% sugar solution placed on the screen top).
Be careful not to suck or blow too hard, as mosquitoes are fragile.
Do not collect more than five mosquitoes in the sucking tube
Fig. 3.8 collection using sucking tube

54
ii) Back-pack aspirators
Fig 3.9 Collections using back pack aspirator
Backpack aspirators powered by rechargeable 12-volt batteries have proven to be an efficient and
effective alternative means of collecting resting adult mosquitoes.
collections can be done in both indoors and outdoors. It is a standardized and less labour-
intensive method in and around human habitation.
.It is a standardized and less labour-intensive method.
Following a standardized, timed collection in selected rooms of each house, densities are
recorded as,
1. the number of adults per house (females, males or both) or

2. the number of adults per man-hour
When the mosquito population density is low, the percentage of houses positive for adult vector
mosquitoes is sometimes used.

55
iii) Using Resting Boxes
A small wooden box ( 25cm X 23cm X 40 cm) is used and inside of which is covered with a
black muslin cloth. One end is open. Outside is covered with a black non reflective paper.
The boxes can be placed both indoor and outdoor to collect mosquitoes.
Resting boxes are generally placed on the ground with the open end facing west to
minimize the influence of direct sunlight during the early part of the day.
For surveillance purposes, resting boxes are usually positioned no closer than 10 ft from
one another in either a line or grid design.
Personnel making collections from resting boxes should be equipped with catch covers,
sprayers, and mechanical aspirators. Catch covers are squares of heavy cloth, similar in
design to a shower cap, and are used to cover the boxes and trap the resting mosquitoes
during the collection process.
Collection involves approaching the box from the rear, slipping the catch cover over the
open end of the box, and spraying short bursts of anesthetic through the screened hole to
anesthetize the mosquitoes trapped inside.
The mosquitoes are collected by removing the catch cover and aspirating the specimens
directly from the box with a mechanical aspirator.
Studies have shown that use of resting boxes to conduct resting collections is a practical
method for use in routine sampling of Aedes mosquitoes .
Fig. 3.10 collection using resting boxes

56
3.7 Sampling the ovipositing population
3.7.1 Oviposition traps
These devices, also known as ovitraps, constitute a sensitive and economical method for
detecting the presence of Ae. aegypti and Ae. albopictus in situations
where infestations are low and larval surveys are generally unproductive (e.g. when the
Breteau index is <5).
Specially useful for the early detection of new infestations in areas from which the mosquito has
been eliminated.
Useful for surveillance at international ports of entry which, in accordance with international
sanitary codes, should be kept free of vector foci.
Fig 3.11 Oviposition traps
3.7.2 Standard ovi Trap
The standard ovitrap is a wide-mouth 0.5 litre glass jar painted black on the outside.
A hardboard or wooden paddle is clipped vertically to the inside with its rough side facing
inwards.
The jar is partially filled with clean water and is appropriately placed in a rain-sheltered site
usually outdoors and close to habitation.
Ovitraps are usually serviced every other day and the paddles are examined for the presence of
Ae. aegypti eggs.
The percentage of positive ovitraps provides the simplest index of infestation levels.
In more detailed studies, all the eggs on each paddle are counted and the mean number of eggs
per ovitrap is calculated.
Ovitraps with plant germination paper as a substrate for egg deposition can also be used.

57
Fig 3.12 Ovi trap with a paddle
For accurate interpretation, field records must indicate the location of each ovitrap and its
condition at the time of servicing. If a trap is flooded, dry, missing, or overturned, the data
should be discarded.
Ovitraps are inexpensive and it is possible to install and service them over large areas
relatively quickly.
They can also be used by people without specialized training.
a) Enhanced CDC ovitrap
More attractive to ovipositing females and yields many more Ae. aegypti eggs than the
standard version.
In this double ovitrap method, one jar contains an olfactory attractant made from a
standardized 7-day-old hay infusion, while the other contains a 10% dilution of the same
infusion.
The enhanced ovitrap has proved suitable for monitoring changes in the adult female
populations daily rather than weekly and has been successfully used to assess the impact of
adulticidal space spraying on adult females.
Alternatives to hay infusions have also been used to improve the attraction of standard
ovitraps.
While ovitraps can be used to monitor changes in oviposition activity over time, comparisons
between areas are not reliable because the availability of larval habitats in which females can choose to
lay eggs will differ. Similarly, it can be misleading to monitor and interpret ovitrap data over a time in a
given area where vector control interventions include source reduction measures.

58
b) Sticky trap collections
Various sticky trap devices have been used for sampling adult Ae. aegypti. They may be designed to be
visually attractive, odour-baited, or both, or are simply located at constricted access points through which
adult mosquitoes pass (e.g. at points of exit and entry from subterranean habitats such as keyholes in
service manhole covers in roads). Age and viral infection have been determined in adult mosquitoes
collected with sticky traps though mainly in a research context.
3.8 Data Recording
Data management is an important part of any surveillance program. Therefore, correct data recording is
fundamental.
The basic information collected with each sample should be
1) The date
2) location or site
3) type of habitat
4) climatic conditions (temperature, relative humidity, rainfall)
5) No. of houses/premises examined.
6) No. of houses found with breeding places.
7) No. of houses found with adult mosquitoes.
8) The larval or pupal density, stages present and the species (determined in the lab through
identification).
9) No. of wet and dry containers
This represents the basic information to be collected; if program managers/planners wish to include
more than that, of course, it is their prerogative. The data recording formats are given in the annexures.
3.9 Monitoring environmental and social risks
In addition to the evaluation of aspects directly pertaining to vector densities and distribution,
community-oriented, integrated vector management strategies require that other parameters be measured
or periodically monitored.

59
Various factors have been determined to influence a communitys vulnerability to dengue epidemics.
The distribution and density of the human population,

Settlement characteristics,
Conditions of land tenure,
Housing styles, education and
Socio economic status
Water drainage, water disposal
These factors are all interrelated and fundamentally important for planning and for assessing
dengue risk.
Knowledge of changes in the distribution of water supply services and their quality and reliability
over time, as well as knowledge of domestic water storage practices and solid waste disposal services, are
of particular relevance.
This type of information helps in establishing ecological profiles that can be of value for planning
targeted source reduction or management activities and for organizing epidemic intervention measures.
Some of these data sets are generated by the health sector, while others are derived from external
sources. In most cases, annual or even less frequent updates will suffice for programme management
purposes.
However, in the case of meteorological data, especially rainfall patterns, a more frequent analysis
(such as weekly or monthly) is warranted if the data are to be of predictive value in determining seasonal
trends and short-term fluctuations of the vector population.

60
Annexure 1
Daily Return of Dengue Vector Surveillance in . District
MOH area . Locality/Road

Date
No.of containers Presence of Aedes spp.

Type of breeding
Name & address
No. of Aedes
Presen-ce of
other mosq.
holder /public
of the house
Examined
albopictus
pupae
Aedes spp
for Aedes
Remarks
Serial no.
Without
Positive
spp
aegypti
(A&B)
source
Other
water
water
Aedes
Aedes
Aedes
With
Both
spp.
site
(A)
(B)
Id Od Id Od Id Od Id Od Id Od Id Od Id Od Id Od Id Od Id Od
...
61
Name of Entomological Assistant Signature of Entomological Assistant
Annexure 2
Summary of Daily Dengue Vector Surveillance in .. District
MOH area Locality/Road
Date
Nature of the locality :- Residential Area rural/ Residential Area -urban/Town area/Public site
Climatic conditions :- Weather Bright sun shine/Gloomy/Raining heavily/Drizzling
Temperature ..0C Relative humidity ..
Reason for the Survey:- Sentinel site / Report of a dengue case / Request from a MOH / Other
Type of No. of No. of No. of No. of Pupal No. of

breeding containers containers containers containers Index containers
source with water without Positive for Positive for Positive
water Aedes Aedes pupae for other
mosquito
spp.
Id Od Total Id Od Total Id Od Total Id Od Total
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
Name of EA. Signature of EA...

62
Annexure 3
Abbreviations for Vector Breeding Sites
Abbreviati
Description Abbreviation Description
on
WSGT Water Storage Ground Tanks WSOHT Water Storage Over Head Tanks
RWHT Rain Water Harvesting Tanks WSPST Water Storage Plasti Shell Tanks
WSFGT Water Storage Fibre Glass Tanks WSGT Water Storage Glass Tanks
WSPB Water Storage Plastic Barrels WSPC Water Storage Plastic Containers
WSEW Water Storage EarthenWare WSMC Water Storage Metal Containers
WSMB Water Storage Metal Barrels Dis.GW Discarded Glass Ware
Dis.PW Discarded Plastic Ware Dis.MW Discarded Metal Ware
Dis.RW Discarded Rigiform Ware Co.Shell Coconut Shells
Dis.Shoe Discarded Shoes NU.Tyre Non Used Tyres
NU.Well Non Used Wells NU.Boat Non Used Boats
Mch.Part Machinery Parts Po.Sheet Polythene Sheets
Po.Bag Polythene Bags Ce.Slab Cement Slabs
Rf.gut Roof Gutters AC.Tray Air Condition Trays
Or.Pond Ornamental Ponds Or.WC Ornamental Water Containers
B.Bath Bird Baths FP.Plt Flower Pot Plates
FP.Hrd Flower Pots with Hardened soil Earth P Earth Pipes
Mon. Monuments Ce.Drain Cement lined Drains
Dr.Tank Drop Tanks (in irrigation canals) Ce.Ir. Cn Cement lined Irrigation Canals
Veg.Pt Vegetation Parts (fallen on ground) Bmb.St Bamboo Stumps
Tr.Hole Tree Holes Le.Axl Leaf Axils

63
Fl.Vas Flower Vases Ref. Tr Refrigerator Trays
Ant.Tr Ant Traps NS.Com Non Used Comodes
NS.cist Non Used Cisterns Gul.Tr Gully Traps
Any natural breeding place other Any man-made breeding place

Other -1 Other -2
than specified other than specified

64
Annexure 4
..
..
..
20.././
Medical Officer of Health

...
REPORT ON DENGUE VECTOR SURVEILLANCE
Date : .. Type of survey :
GN area : .. Locality :
Reason for the Survey:- Sentinel site/Report of a dengue case/Request from a MOH/ Other
Name of the patient
Address:...
Findings:
(1).
Premise (House) Index(%) Container Index (%) Breteau Index (%)

Pupal
Aedes Aedes Common Aedes Aedes Common Aedes Aedes Common Index
aegypti albopictus index aegypti albopictus index aegypti albopictus index
For control purposes consider the Indices combined for both species.
Data were collected through a random sample of . premises

65
% Premises found with (potential) % Premises with wet Most prevalent potential
dry breeding sites containers breeding places
Comments of the E.A
i) Premises for which special attention has to be paid
1. 2
3. 4
ii) Premises with fever patients
1. 2. .
iii) Other comments -

.
..
Recommendations of Entomologist/ RMO:
Make the people aware to conduct the below mentioned Vector Control (VC) methods and follow up
frequently.

66
Annexure 5
Recommen- Recommen-
Vector Control method Vector Control method
dation dation
1 Introduction of larvivorous 2 Scrub the inner sides of Water

fish in to Water storage tanks storage tanks / Water storage
& ornamental ponds containers/ at least once a
week before refilling water
3 Proper covering of WSC with a 4 Use of Abate 1% SG for Water

tight lid storage tanks
5 Use of IGR for Water storage 6 Keep non used tyres under a
tanks/Rain water harvesting shelter to avoid the collection
tanks/blocked drains of rain water
7 Filling sand into tyres 8 Puncturing the tyres
9 Root out the unnecessary 10 Scrub the inner sides of the

water collecting plants bird baths /ornamental water
(vegetation) containers at least once a
week & refill water
11 Scrub the inner sides of animal 12 Weekly introduction of Bti in

feeding containers at least Water storage tanks Rain
once a week & refill water water harvesting tanks
/blocked
drains/vegetation/cement
slabs/ dumping grounds
13 Remove or berry water 14 Clean roof gutters

containing vegetation parts
15 Put Abate 1% SG in roof 16 Cover overhead tanks

gutters properly
17 Cover the overflow tubes of 18 Cover the openings of the rain

over head tanks with a piece water harvesting tanks
of cloth properly
19 Remove discarded items in a 20 Turn non used boats upside

proper way (destroy, berry down or keep under a shelter

67
,burn or send to a recycling

centre)
21 Keep machinery parts under a 22 Add soap or salt into ant

shelter traps/refrigerator trays
22 Organize cleaning up 24 Introduce larvivorous fish into

campaigns abandoned wells
:- Recommend :- Highly Recommend - :- Not necessary now
Remarks
..
..
Entomologist/ Regional Officer /AMC
(Your feedback would be greatly appreciated).

68
Chapter 4
Dengue Vector Control
Introduction
Dengue vector mosquitoes use a wide range of confined larval habitats, both man-made and
natural. Some man-made container habitats produce large numbers of adult mosquitoes, whereas others
are less productive. Thus, control efforts should be targeted at the most productive and hence
epidemiologically more important larval habitats, especially when working with limited resources. Such
targeted strategies require a thorough understanding of the local vector biology, bionomics and ecology
and good knowledge, attitudes and practices of the community towards dengue vector control.
Dengue vector control measures include
1. Environmental management
2. Biological control
3. Chemical control
4. Genetic control methods
5. Personal protection methods
4.1 Environmental management
Environmental management is changing the environment to prevent or minimize vector propagation and
human contact with the vector-pathogen by destroying, altering, removing or recycling of containers that
provide larval habitats. This has two components viz. (4.1.1) environmental modification and (4.1.2)
environmental manipulation.
4.1.1 Environmental modification - long lasting or permanent physical changes of vector habitats
Examples
Mosquito proofing of overhead tanks, rain water harvesting tanks , underground storage
tanks, domestic wells or masonry chambers of piped waterlines
Proper drainage of cement lined or concrete drains
Proper disposal of discarded receptacles (burrying, crushing, burning etc.)
Removal of unnecessary water collecting plants like Bromelia
Mosquito proofing of wells by closing them with cement slabs and installing hand pumps

69
4.1.2 Environmental manipulation - measures that have a temporary effect on vector habitat and need
to be repeated.
Examples
Covering of water storage containers or tanks with tight lids, screens or net covers
Removal of water with scrubbing and cleaning of inner surfaces of water storage
containers, tanks or flower vases at least a once a week
Changing water salinity and pH in ant traps, refrigerator trays etc. by adding salt or soap
Periodic inspection and cleaning of roof gutters, sunshades and concrete slabs of the
buildings
Puncturing blocked flower pots to produce a drain hole
Keeping machinery parts and scrap material of factories in a sheltered place to prevent
collection of rain water
Proper tyre management, storing un-used tyres under a shelter in bus depots and other
places
Un -used boats and canoes should be kept upside down.
Fig 4.1 Covering of breeding sites

70
4.1.3 Use of Expanded polystyrene beads
Use of expanded polystyrene beads on the surface of water provide a physical barrier that inhibits
oviposition by female mosquitoes in water storage containers. Water of these containers can be drawn
from below via a pipe, thus, preventing overflow of the beads. These beads can also be used in septic
tanks which Ae. Aegypti sometimes exploits.
Covering of water surface completely with a material that is impenetrable to mosquitoes

is a less conventional approach for mosquito control.
Application of a layer of polystyrene beads prevents mosquito larvae from reaching the
water surface to breathe. Thus, mosquito larvae die because they are unable to breathe.
The beads do not decay and remain floating for years.
Because the beads are easily blown or washed away, they can be used only in confined
water bodies.
Polystyrene beads are not toxic to humans, animals or fish and are safe for use even for
drinking-water.
When polystyrene beads are used, the beads are expanded by heating them to 100 C in
steam or boiling water before the application into breeding sites.
Expanded polystyrene beads can be spread on water to form a floating layer. A layer of
12 cm thickness is sufficient to prevent mosquito breeding, if it covers the surface
completely.
Fig. 4.2 use of polystyrene beads

71
4.1.4 Improvement of water supply and water-storage systems
Improving water supply is a fundamental method of controlling Aedes vectors, specially Ae.
aegypti.
Reliable supply of potable water prevents water-storage in containers such as drums, overhead
and/or ground level tanks and barrels and concrete jars that serve as larval habitats
In urban areas, use of cost-recovery mechanisms such as the introduction of metered water may
encourage household collection and storage of roof catchment rainwater that can be harvested at
no cost, resulting in the continued use of storage containers.
Traditional water storage practices may also persist even when reliable supplies are available.
The installation of reliable piped water supplies in houses should therefore be accompanied by a
communication strategy that discourages traditional storage practices.
Water piped to households is preferable to water drawn from wells, communal standpipes,
rooftop catchments and other water-storage systems.
4.1.5 Mosquito-proofing of water-storage containers
Water-storage containers can be designed to prevent access by mosquitoes for ovi position.
Containers can be fitted with tight lids or mosquito proof net covers or, if rain-filled, tightly-fitted
mesh screens that allow for rainwater to be harvested from roofs while keeping mosquitoes out.
Removable covers should be replaced every time when water is removed and should be well
maintained to prevent damage that permits mosquitoes to get in and out.
4.1.6 Solid waste management
In the context of dengue vector control, solid waste refers mainly to non-bio degradable items
of household, community and industries. Reducing the amount of solid waste in the environment
substantially reduce the availability of Aedes larval habitats.
Proper storage, collection and disposal of waste is essential for protecting public health. The basic
rule of reduce, reuse, recycle is highly applicable.
Efforts to reduce solid waste should be directed against discarded or non-essential containers,
particularly if they have been identified as important mosquito breeding sites in a particular area.
Solid waste should be collected and disposed of regularly. The frequency of collection is
important at least once a week.
It is important to inform the community about waste disposal in order to encourage them for
proper waste disposal.
Containers that can be recycled that make profit for both small and large-scale businesses.
Used tyres are common and highly productive larval breeding habitats that needs special
attention.

72
Discarded tyres should be collected, recycle or disposed of by proper incineration in waste

transformation facilities (e.g. incinerators, energy-production plants, or lime kilns fitted with
emission control devices).
Tyres can also be recycled in a variety of ways, including for use as shoe soles, flooring,
industrial rubber gaskets or household hardware (e.g. buckets, rubbish bins).
Industrially shredded tyres can be incorporated into road surfacing materials. Sanitary regulations
may require that whole tyres are buried in a separate area of a landfill to avoid their rising
upwards under compaction and disrupting soil cover.
Regulation of the sale of new tyres mandating the payment of an additional deposit and return
charge may also be an incentive for better management and disposal of old tyres.
4.1.7. Street cleansing
A reliable and regular street cleansing system that removes discarded water-bearing and dry
containers and cleaning drains to ensure they do not become stagnant and breed Aedes mosquitoes will
help to reduce larval habitats
4.1.8. Building structures
Buildings with water holding structures (roof gutters etc) and construction sites have been
identified as high risk sites for Ae. aegypti and Ae. albopictus breeding. Thus, during the planning and
construction phases of buildings and other infrastructure, including urban renewal schemes, opportunities
arise to modify or reduce potential larval habitats possibly through legislation and regulation
For example, under revised legislation in Singapore, roof gutters are not permitted on buildings in
new developments because they are difficult to access and maintain. Moreover, property owners are
required to remove existing gutters on their premises if they are unable to maintain them satisfactorily.
4.2 Biological Control
Biological control is based on the introduction of organisms that prey upon, parasitize, compete
with or otherwise reduce populations of the target species
Certain species of larvivorous fish and predatory copepods (Copepoda: Cyclopoidea) small fresh
water crustaceans have proved effective in specific container habitats.
Use of biological agents would avoid chemical contamination of the environment.
However, there may be operational limitations such as the expense and task of rearing the
organisms on a large scale, difficulty in applying them and their limited utility in aquatic sites where
temperature, pH and organic pollution may exceed the narrow requirements of the organism.
Effective only against the immature stages of vector mosquitoes in the larval habitat where they are
introduced.

73
Biological control organisms are not resistant to desiccation, so their utility is mainly restricted to
container habitats that are seldom emptied or cleaned, such as large concrete or glazed clay water-
storage containers or wells.
The willingness of local communities to accept the introduction of organisms into water containers is
essential.
Community involvement is desirable in distributing the fish or copepods, and monitoring and re-
stocking containers when necessary.
Thus, these limitations needs to be overcome when embarking on a biological vector control
programme.
4.2.1. Use of Larvivorous fish
A variety of fish species eat mosquito larvae and thus have the potential of eliminating
mosquitoes from some containers such as containers that are used to store potable water (water storage
tanks, barrels etc.) in many countries, and in open freshwater wells, concrete irrigation ditches and
industrial tanks.
Important larvivorous fish species that can be used for dengue mosquito larval control are
Top minnow or Nalahandaya(Aplocheilus spp.) is most efficient in clean water.

Guppy (Poecilia reticulata) can be used successfully even in organically polluted water.
Tilapia spp (Orechromis mossambicus and O. nilotius) juvenile stages are highly voracious
and very effective in mosquito larval control
The viviparous species Poecilia reticulata adapts well to these types of confined water bodies and has
been most commonly used. Use of native larvivorous fish is preferred as exotic species may escape into
natural habitats and threaten the indigenous fauna.
Fish can be transported from a breeding pond to a

place where they are needed in half water filled
(preferably oxygenated) plastic bags.
Fig 4.3 Adding larvivorous fish to breeding site

74
4.2.2. Use of Bacteria
i) Bacillus thuringiensis israelensis (Bti)
Bt.H-14 is a naturally occurring organism and scientists isolate it from soil, insects and plant
surfaces.
First discovered in 1977 in the Central Negev Desert of Israel. Since its discovery , the
organism has shown rapid larvicidal activity against both mosquitoes and black flies
Bt has a highly specific mode of action against a narrow host spectrum. This selective activity
is limited to the Order Diptera and specifically to families within the suborder Nematocera to
which Ae. aegypti and Ae. albopictus belong. It is an environmentally non-intrusive mosquito
larvicide.
Evaluations conducted on mammalian toxicity and pathogenicity have shown that there is no
evidence of multiplication of the organism in mammals.
However, effect of Bt on human health and the environment depend on how much Bt is
present, the length and frequency of exposure and the health of a person or certain
environmental factors.
The Bt products that are recommended by the WHO are safe for humans when used in
recommended dosages.
Bt.H-14 is commercially available under different trade names
ii) Mode of action of Bt
The active ingredient is composed of viable endo spores and Delta-endotoxin crystals.
Larvicidal activity is dependent upon the delta-endotoxin crystals which must be ingested by
the mosquito larvae.
Upon ingestion , pH conditions, and enzymes in the gut of the larvae rapidly hydrolyze the
protoxin into active sub units which attack the mid gut of larvae.
General gut paralysis occurs within a few hours and activity ceases. The cells of the larval
midgut , particularly those in the region of posterior stomach and gastric caecae become
extensively damaged.
Disruption of ionic regulation capacity of the midgut, and subsequent flow of toxic
substances into the larval haemocoel causes death within 24 hours.
Slow release formulations of Bt. H-14 are being developed. Briquette formulations appear to have a
greater residual activity. Bt. H-14 formulations tend to rapidly settle on the bottom of water containers
and therefore frequent applications are required.

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4.2.3.Other biological control Agents
i) Predatory copepods
Various predatory copepod species have also proved effective against dengue vectors in
operational settings.
However, although copepod populations can survive for long periods, as with fish, re-
introductions may be necessary for sustained control.
A vector control programme in Northern Viet Nam using copepods in large water-storage tanks,
combined with source reduction, successfully eliminated Ae. aegypti in many communities and
has prevented dengue transmission for a number of years. To date, these successes have not been
replicated in other countries.
Fig 4.4 Mesocyclops Fig 4.5 Dragon fly nymph

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Fig 4.6 Predatory mosquitoe larvae - Toxorhynchitis
ii) Nematodes - Ramanomermis

iii)Entomopathogenic fungi - Coelomomyces
iv)Neem - an oil extract of seeds of the neem tree,
Azadirachta indica, which has larvicidal properties;
4.3 Chemical Control
i) Larval control
ii) Adult control
4.3.1. Larval control
In dengue vector control, larvicides are usually used in domestic mosquito breeding containers or
tanks that cannot be destroyed, eliminated or where other mosquito control methods are
impossible or too expensive to use. It can also be used in cement or concrete lined drains where
drainage is difficult.
Application of chemical larvicides on a long-term basis is difficult and expensive. Therefore
chemical larvicides are best used in situations where the disease and vector surveillance indicate
the existence of certain periods of high risk and in localities where outbreaks might occur. Thus,
establishing proper timing and location are essential for maximum effectiveness of chemical
larviciding.

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Although chemical larviciding is carried out in selected locations under transmission risk
conditions, the house occupants should be encouraged to control larvae by environmental
sanitation
Chemical larviciding is also justified where the density of the human population to be protected
is sufficiently high.
Use of chemicals for larviciding in dengue control face problems due to development of vector
resistance to insecticides, high cost and detrimental effects to the environment
4.3.1.1 Chemicals that can be used as larvicides in dengue vector control
i) Temephos (Abate)1% Sand Granules
Abate (Temephos) is an organophosphate insecticide effective against the aquatic larval stages of
some insects. It causes a rapid death of Aedes larvae
Because of its relative safety to mammals and birds it has been widely used for control of
mosquito larvae since 1965.
In many parts of the world Abate has been approved for the use in drinking water supplies for the
control of mosquitoes and guinea worms.
The manufacturer and WHO recommend application of temephos 1% SG at the rate of 1g/10 litre
of water is safe for drinking water. Thus, it is recommended the use of temephos 1% SG for water
storage containers at the said dosage as a supplementary measure for dengue larval control in Sri
Lanka.
It is very important to make every effort not to exceed the permitted dose when the product is
applied to water that may be used for drinking and cooking purposes.
Note: Organophosphates are nerve poisons which kill by inhibiting the action of certain enzymes
(Cholinesterase) in the nervous system.
a) Selection of sites
Temephos 1% SG should be used only in containers where water is likely to be stored for a
period at least one week or more. It is most applicable to use in domestic and peridomestic water
storage receptacles and cement tanks that have been identified as breeding sites for Aedes aegypti
and Ae. albopictus mosquitoes.
b) Dose for application

The recommended dose of application is 1g of product per 10 liters of water (1 ppm of active
ingredient). Application should be carried out assuming that the storage of water in the tank is
maximal or the usual storage volume, even if the tank is not fully filled with water.
Advise the occupants to fill the tank to the maximum level and not to use the water for drinking
purposes when it is not filled up to the maximum level.

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c) Application frequency
Temephos 1% SG should be applied 3-4 times a year based on the prevalence of dengue vector
mosquitoes in the area.
ii). Use of Insect growth regulators (Pyriproxyfen)
These are chemical compounds which prevent the development of larvae into adults. It does not
have undesirable effects on man, wild life and the environment and compatible with modern insect pest
management principle. In general IGRs have long residual effects (three to six months) at relatively low
dosages .
There are two types of growth regulators.
Juvenile hormone analogues
It prevent the development of larvae into viable pupae.
Eg: Pyriproxyfen, Methoprene ,Fenoxycab
Chitin synthesis inhibitors - interfere the moulting process.
Eg: Dichlorbenzuron, Diflubenzuron, Trif lumuron
a) Selection of sites
Better to use in places like water storage containers including rain water harvesting tanks.
b) Dose for application
The recommended dose of application is 2g of product per 1000 liters (1 cubic meter) of water .
Application should be carried out assuming that the accumulation of water in the container/tank/drain is
maximal or the usual storage volume , even if the water body is not fully filled with water.
Pyriproxyfen should be used only in places where water is likely to be accumulated or stored for a period
more than one week .

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4.3.2 Adult control
i) Space-spraying with insecticides
Insecticidal aerosols are used for the killing of flying and resting mosquitoes during outbreaks of
dengue fever or at high densities of Ae. aegypti/ Ae. albopictus in areas where there is potential risk of
dengue transmission. Space sprays are usually applied in and around houses in cities or villages and
sometimes on outdoor resting places in dense vegetation. Space spraying is carried out using special
equipment such as motorized knapsack mist-blowers, shoulder-carried thermal foggers, vehicle- or
aircraft- mounted aerosol generators. Space-spraying involves the use of thermal or cold fogs and ultra-
low-volume sprays. Because the insecticidal action does not last long it is usually necessary to repeat the
procedure several times at pre designed time intervals.
Thermal Fogs - In thermal fogs, insecticide is vaporized by a high velocity stream of hot gas
to make droplets. Droplet size of thermal fogs is < 15 microns.
ULV Cold Fogs - Application of minimum quantities of liquid-concentrated insecticides.

The droplets are formed by mechanical breaking up of the spray mixture.
i). Space spraying Indoor
Fig 4.7 Space spraying Indoor

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ii). Space spraying Outdoor
Fig 4.8 Space spraying - Outdoor
Fig 4.9 vehicle mounted fogging machine

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i) Pressurized spray cans

Pressurized cans provide a convenient method of spraying insecticidal aerosols in rooms, on
mosquito nets, vehicles and so on, to obtain rapid knock-down of mosquitoes. Pyrethrum used to be the
common ingredient in many different brands of aerosol sprays.
Space sprays have a very short residual effect: the active ingredients are rapidly degraded by light. An
advantage of short-lasting insecticides is that they do not leave any toxic residues on beds, furniture or
other surfaces. This method works best in screened spaces and can be repeated daily or several times a
day. Aerosol sprays containing rapidly acting insecticides are used for immediate killing of flying
mosquitoes.
Fig 4.10 Use of pressurized can
4.4 Genetic Control Methods
Sterile male technique the release of sterilized males (by ionizing irradiation or chemical
compounds). Sterilized males seek out and mate with the wild females in natural population thus
preventing the hatching of their eggs.

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4.5 Personal Protection methods
Reduce manvector contact, such as installing mosquito screening on windows, doors and other
entry points, and using mosquito nets while sleeping during daytime.
The choice of approach should be effective, practicable and appropriate to local circumstances.
4.5.1 Making houses and shelters insect-proof
a) Anti-mosquito screening
Screening of doors, windows and other openings in houses prevents entering of mosquitoes into
houses, while maintaining some ventilation. To stop entering mosquitoes into houses, the holes of the
netting material should be 1.5 mm or less. Screening is not much popular because of the restriction on
ventilation.
b) Insecticide-treated screening and curtains

Treated screening and curtains are as effective as mosquito nets.
Fig 4.11 Fixing screens
Screening material
Cotton netting: efficient but easily damaged: ventilation is reduced by upto 70%.

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Metal Screens: ventilation is reduced by 30% -50%. Rodents are prevented from entering. Many metal
corrode rapidly in humid areas. Stainless steel or copper screens avoid this problem but are expensive.
Plastic screens : Cheap and easily fitted. Ventilation is reduced by up to 35%. Nylon screening is not
durable when exposed to direct sunlight. Fibre-glass coated in PVC is more durable.
4.5.2 Use of Repellents
Repellents are among the most commonly used methods to prevent mosquitoe bites. They are
applied directly to the skin or to clothing and other fabrics such as bed nets and anti-mosquito screens.
Repellents evaporate much more quickly than most insecticides. The duration of protection by a repellent
applied to skin may range from 15 minutes to 10 hours and on clothing and other fabrics the effect lasts
much longer.
The effectiveness and duration of the repellent depend on
- the type of repellent

- the mode of application,
- local conditions (temperature, humidity wind),
- the attractiveness of individual people to insects,
-loss due to removal by perspiration and abrasion
- and the sensitivity of the insects to repellents,
Repellents should not be sprayed on the face. The eyes or mucous membranes (nose, mouth)
should not be treated. Instead, they can be applied by spraying on to the hands and then rubbing on to the
less sensitive parts of the face as necessary. If an allergic skin reaction is observed, the treated skin should
be washed with water and consult a physician.
4.5.3 Protective clothing
Clothing can offer protection from mosquito bites when it is of correct thickness and texture
through which the mosquito cannot easily bite. Lighter colours generally attract fewer insects than darker
colours. Thick socks, boots in combination with long trousers, long sleeved shirts, head nets, collars and
hats offer protection against mosquito bites.

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4.5.4 Treated clothing
Clothing can be treated with repellents to prevent insects from landing or feeding, or with quick-
acting insecticides of the pyrethroid group, such as permethrin. These latter compounds do not repel the
insects but allow them to make contact with the fabric and irritate or kill them before they manage to feed.
Repellents may remain effective for up to a week when applied to clothing. An extended efficacy
can be obtained by sealing the impregnated fabric in a container or airtight bag when not in use to prevent
evaporation of the repellent.
i) Jacket ii) Head nets
iii) anklets
Fig. 4.12 (i), (ii), (iii) various impregnated clothes

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4.5.5. Use of Mosquito nets
i) Mosquito nets
Plain mosquito nets acts as a mechanical barrier
ii) Insecticide-treated mosquito nets (ITN) and other materials
Types of Insecticide Impregnated Bed Nets
a) Conventionally Insecticide Impregnated Nets

b) Long Lasting Insecticidal Nets - (LLINs) are ready-to-use pre-treated mosquito nets, which
require no re-treatment during their expected life span 4-5 years
Nets treated with pyrethroids give greater personal protection than untreated nets by irritating,
repelling or killing mosquitoes before they can find a place to bite through the net.
The presence of one insecticide treated mosquito net in a room may also partially protect individuals
sleeping outside the net.
Table 4.1 WHOPES-recommended insecticide products for treatment of mosquito nets
Dosage
Insecticide product
(Active ingredient in mg/m2 of netting)
Alpha-cypermethrin 10%SC 20-40
Cyfluthrin 5%EW 50
Deltamethrin 1%SC and WT25% 15-25
Etofenprox 10%EW 200
Lambda-cyhalothrin 2.5%CS 10-15
Permethrin 10%EC 200-500
EC = emulsifiable concentrate; GR = granule
The dosage will depend on the formulation used.

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4.5.6 Bed curtains
In areas where bed nets are too expensive an alternative may be, to use curtains made of locally
available fibres or strings hung around the bed. These open curtains must be treated with an insecticide
to get protection from flying insects.
4.5.7 Treated bed sheets
People sleeping out of doors, where mosquito nets are unaffordable or impractical, could consider
covering themselves with sheets or other fabrics treated with insecticide or repellent. This method has not
yet been tested but it can be expected to be as safe and effective as use of treated clothing.
i) Treating fabrics with an insecticide
The impregnation of fabrics with an insecticide is simple: an emulsion is made in water, and the
material to be impregnated is soaked in it and allowed to dry. After drying, the insecticide remains
attached to the fibres.
ii) Alternative materials for screening:
Instead of gauze, it is also possible to use fibres and stripes or loose hanging curtains for treatment.
Materials that can be used
Fibres obtained by unpicking polythene or jute sacks.

String
Bead curtains
Plastic strips
4.5.8 Treated sheeting for temporary shelters
Insecticide-treated sheeting offers a more lasting solution. This material is attached to the poles of
the shelter which support the roof, and can also be used to cover door and window openings.
It can be rolled up during not in use. Some mosquitoes that rest outside or inside on the sheeting
are killed, and others are repelled after brief contact.
The material must be strong, cheap and suitable for treatment. Woven polypropylene meets these
requirements and is widely available. The pyrethroid insecticides appear to adhere well and show good
resistance to being washed off by rain

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Insecticide treated sheeting of woven polypropylene can be attached to the poles of temporary
huts
Fig 4.13 Treated sheeting for temporary huts
i) Treatment method
The sheeting can be soaked or sprayed with pyrethroid insecticides, following the instructions given
on p. 86. For speed and convenience, spraying may be preferred where spray pumps are available.
Recommended dosages per square metre are 0.75 g of permethrin, 0.05 g of cyfluthrin, or 0.025 g of
deltamethrin or lambdacyhalothrin.
4.5.9 Mosquito coils / vaporizing mats
Coils and mats are among the most popular and widely used insecticide vaporizers because they
are easy to use, effective and inexpensive.
Where electricity is available, small electric heating plates can be used to vaporize volatile
insecticides from mats. This popular method has the advantage over coils that no visible smoke is
produced.
4.5.10 Electric liquid vaporizer
This device is a technological improvement on vaporizing mats. The insecticide is evaporated by

an electric heater through a porous wick from a reservoir bottle containing the liquid.

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4.5.11 Avoidance and diversion of biting Diptera
Personal protection is sometimes possible by avoiding places where mosquitoes and biting flies
are known to rest or breed, and by not visiting risky places during peak biting hours. For many species,
these are the hours immediately after sunset and before sunrise.

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4.6 Insecticide Susceptibility test for Adult Mosquitoes
4.6.1 Objectives:
The purpose of the susceptibility test is to detect the emergence of resistant individuals in a
mosquito population as early as possible before it is widely established. For this purpose it is necessary;
a) To establish the base-line susceptibility of a normal populations of mosquitoes

b) To expose mosquito populations to discriminative dosage of insecticide at periodic
intervals to detect any tolerant individuals and to monitor any changes in their susceptibility
levels
a) Establishing the base-line susceptibility of normal population
Normal population means a population never subjected to insecticide pressure in which resistant
individuals are rare. Batches of mosquitoes are exposed to different concentrations of the relevant
insecticide for 60 minutes. Mortality is determined after 24 hour recovery period. The concentrations
should be chosen such that at least one concentration gives 100% mortality, some give 50%-90%
mortalities, and at least 2 concentrations give mortalities between 5%-50%. Base-line data is plotted on
logarithmic probability paper to determine the concentration that gives 99.9% mortality. Double this
concentration is taken as the discriminating or diagnostic concentration.
b) Monitoring of susceptibility levels by routine checks using discriminating concentration
4.6.2 Monitoring of susceptibility levels in Adult mosquitoes
Requirements
i) Insecticide impregnated papers
The insecticide papers with the discriminating concentrations of insecticides and control papers are
packed in plastic boxes each contains 8 papers. The discriminating concentrations of insecticides are
given in table 4.2.

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Table 4.2 Discriminating concentrations of insecticides for Aedes vectors
Insecticide Class Insecticide Concentration
Organochlorine DDT 4%
Organophosphate Malathion 0.8%
Carbamate Propoxur 0.1%
Pyrethroid Permethrin 0.25%
Lambdacyhalothrin 0.03%
Deltamethrin 0.025%
ii) Conditions of mosquitoes
Females should be used. It is recommended ideally the tests should be undertaken on non blood fed adult
females of known age (24-48 hrs post emergence). Mosquitoes of known age can be obtained using larval
collections or the F1 progeny from adult females.
Where adults obtained from larval collections are used, the type of breeding site ( i.e. water
storage tanks, tyres, leaf axils etc.) should be specified.
Where only adult females can be used, their physiological status ( i.e. unfed, blood fed, semi-
gravid, gravid) should be recorded.
iii) Conditions of test
The experiments should be done indoors, if possible the buildings free from insecticidal
contamination and extremes of temperature, humidity, illumination and wind.
Temperature and relative humidity should be recorded during the test (both the exposure and the
holding periods). Ideal temperature for testing is 252C. (Never at temperature higher than
30C). Relative humidity should be 70-80%

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iv) Test Kit
WHO standard susceptibility test kit for adult mosquitoes is used.

The components of a test kit as follows:
a) 12 plastic tubes (125mm length and 44mm in diameter), with each tube fitted at one end with 16-
mesh screen. The 12 tubes include:
Five (5) marked with a red dot use as exposure tubes

Two (2) marked with a green dot for use as control tubes
Five (5) with a green dot for use as holding tubes, for pre-test sorting and post-exposure
observation
b) Seven (7) slide-units, each with a screw-cap on either side, and provided with a 20mm filling hole
c) 40 sheets of clean paper (12x15cm) for lining the holding tubes.
d) 14 spring wire clips to hold the papers in position against the walls of the tubes. Of these, the 7
steel clips are for use only for the holding and the control exposure tubes, and the 7 copper clips
are for use in the insecticide exposure tubes.
e) Two (2) glass (or plastic) aspirator tubes of 12mm internal diameter, together with 60cm of
tubing and mouth-piece.
f) One roll of self-adhesive plastic tape
g) Instruction sheets.
v) Test procedure
It is recommended a minimum number of 100 mosquitoes should be tested for any insecticide
concentration, with 4-5 replicates of 20-25 mosquitoes per test. Where possible collect this
number from the same locality and monitored over time to examine the trends.
Test chambers should be kept vertically.
When testing pyrethroids, timed observations of the rate of knock down (kd) should be made
routinely after 10, 15, 20, 30, 40, 50 and 60 minutes of exposure.
Into each of the holding tubes, insert a piece of clean white paper rolled into a cylinder to line the
wall and fasten it in position with a steel spring-wire. Attach the slides to the tube.

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Mosquitoes should be collected in lots, with each individual lot consisting of more than ten
moquitoes who are gently transferred to the holding tubes through the filling-hole in each side.
A pre-test holding period may be necessary to guard against including damaged specimens in the
test. For this purpose, the holding tubes are set upright, screen end up, for 1 hr. At the end of this
time the damaged mosquitoes are removed.
Introduce into each exposure tube a sheet of insecticide impregnated paper, rolled into a cylinder
to line the wall and fastened into position with a copper clip.
Similarly introduce a control paper impregnated with oil alone into control tubes and fasten it in
position with a steel clip.
Introduce the mosquitoes into exposure tubes by attaching it to the vacant screw-top in the slide.
The slide should be pulled out to appoint beyond the filling-hole so that no part of it occludes the
tube openings. The mosquitoes are then blown gently down into the exposure tube. Close the
slide. Detach the holding tube and set it aside.
Leave the exposure tubes standing upright with screen end up for the required exposure period
under conditions of moderate, diffuse illumination and adequate humidity.
At the end of the exposure period, transfer the mosquitoes to the holding tubes by reversing the
procedure. Attach the holding tube, open the slide and gently blow the mosquitoes into the
holding tube. Close the slide and remove the exposure tube. Then set the holding tube so that it
stands on the slide and place a pad of moist cotton-wool on the screen.
Keep the holding tubes for 24 hrs in a secluded, shaded place. If necessary, the tubes should be
protected from ants by placing them on a platform standing in a pan of water. If conditions are
very hot and dry a moist chamber may be prepared by suspending damp toweling in a container.
Mortality counts are made after 24 hours adults should be considered as live if they are able to
fly, regardless of the number of legs remaining.
vi) General remarks
It is recommended that impregnated papers especially pyrethroid papers should not be used more
than 5 times. After the impregnated paper has been removed the package should be resealed
carefully with the plastic tape provided. The paper should be left in the tube, with the open end
well wrapped and placed in the kit box and should be kept in a cool place.

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vii) Interpretation of susceptibility test results
If control mortality is between 5-20%, the average observed mortality should be corrected by
Abbotts formula:
Abbotts formula = % test mortality - % control mortality

X 100
100 - % control mortality
98-100% mortality indicates susceptibility
80-97% mortality suggests possibility of resistance that needs to be confirmed
<80% mortality suggests resistance
4.7 Insecticide Susceptibility test for Mosquito Larvae
In order to detect the emergence of an insecticide resistance strain of a mosquito, it is necessary to

establish a base-line for the species to a given insecticide with samples from an untreated area.
Where regular larviciding operations are used, the normal susceptibility levels of the larvae
should be determined as early as possible. For this several tests should be carried out in different
localities and seasons. Tests should be done at regular intervals to determine the reduction in
susceptibility levels.
WHO standard larval susceptibility test kits could be used
i) Materials required for testing
One (1) pipette delivering 100-1000l

Disposable tubes (100l, 500 l) for measuring aliquots of dilute solutions
Five (5) 1ml pipettes for insecticides and one for the ethanol
Three droppers with rubber suction bulbs
Strainer
Disposable cups or, glass bowls/beakers capacity of 100ml and 250ml
Measuring cylinder
Log-probit papers
Data record forms

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ii) Test Procedure

a) For a complete test with one insecticide, sufficient larvae should be collected from the field. 300
individuals from the same species should be selected; they should be in their 3rd or early 4th instar
and should be retained in the water in which they were collected until selection for testing.
b) Batches of 20-25 larvae should be transferred by means of a strainer to beakers each containing
200ml of water. The average temperature of the water should be 25C.; it must not be below 20C
or above 30C
c) Prepare the test concentration by pipetting 1ml of the standard insecticide solution under the
surface of the water in each of the beakers and stirring vigorously for 30 sec. with the pipette. In
preparing a series of concentrations, the most dilute should be prepared first. Four or more
replicates are set up for each concentration and equal numbers of controls are set up
simultaneously with water in which 1ml ethanol is added.
d) Within 15-30 minutes of the preparation of the test concentration larvae should be added.
e) After 24 hr exposure, larval mortality is recorded. For slow acting insecticides, 48hr reading may
be required. Moribund larvae are counted and added to dead larvae for calculating percentage
mortality. Dead larvae are those that cannot be induced to move when they are probed with a
needle in the siphon or the cervical region. Moribund larvae are those incapable of rising to the
surface or not showing the characteristic of diving reaction when the water is disturbed.
f) Discard the larvae that have pupated during the test. If more than 10% of the control larvae
pupate in the course of the experiment, the test should be discarded and repeated.
g) If the control mortality is between 5-20% the average observed mortality should be corrected by
Abbotts formula
Abbotts formula = % test mortality - % control mortality

X 100
100 - % control mortality

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h) It is important to obtain three mortality counts between 10% -90%. It is not possible to do this
with standard concentrations in the test kit. Therefore, it is necessary to prepare additional
concentrations by diluting a portion of a standard solution with pure ethanol.
General remarks
a) The accuracy of the concentrations provided will be affected if the alcohol is allowed to evaporate
from the standard solutions. Therefore, the bottles should be tightly coppered after use.
b) Test beakers should be carefully cleaned after use to remove traces of insecticides. They should be
thoroughly rinsed, scrubbed with detergent and water. Pipettes should be thoroughly cleaned with acetone
or alcohol.
4.8 Cage Bioassay
Objective:
Caged bioassays are used to evaluate the efficacy of space spraying (fogging) operations.
Space spray are transient and only mosquitoes flying at the time of the application are affected.
The efficacy of space spray is greatly influenced by environmental and operational factors.
Mortality of caged mosquitoes is affected by location of cages (placement), cage design, material and
mesh size. The simple cylindrical cage (20cm length x 5cm diameter) can be constructed by a 16-mesh
nylon material. In the cylindrical cage the two ends are plugged with cork. Make a hole in the lower plug
to introduce mosquitoes.
Test Procedure
Mosquitoes for the bioassay should be obtained from the field collected eggs. Larvae hatched
from the eggs should be reared with an adequate supply of food to ensure uniformity of size.
Shortly before the experiment, (before transfer the mosquitoes to bioassay cages) they should be
fed with a sugar solution.
It is recommended 100 mosquitoes to be tested, with 4-5 replicates. Two to three days old
female mosquitoes should be used for bioassays.

96
Cage bioassays can be performed in different locations (open out door, sites with dense
vegetation, centre in the rooms, rooms with hanging clothes etc.), and different distances.
Thirty minutes after space spray exposure, the cages should be removed and returned to the
laboratory.
Then the mosquitoes are transferred to a simple holding cage, and are provided with sugar
solution and maintained at ambient temperature.
Mortalities are recorded after a 24 hour recovery period. The same number of cages should be
exposed at similar sites in the untreated area as controls.
Temperature and relative humidity should be recorded during the test (both the exposure and the
holding periods). Ideal temperature for testing is 252C. and should not higher than 30C.
Relative humidity should be 70-80%
Chambers should be hung vertically.
Cages should be washed thoroughly before use in another experiment.
Fig. 4.14 Bio assay cage

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4.9 Guidelines for use of chemicals for vector control
4.9.1 Objective of space spraying : To reduce the infected adult vector population and its longevity as
quickly as possible as a supplementary measure for source reduction during outbreaks of dengue.
4.9.2 Organization of the spray team
A spray team should consist of one Public Health Inspector, a PHFO trained for space spraying and three
spray machine operators.
The responsible officer (PHI or PHFO) of the spray unit should be present with the spray unit
throughout the activity for observation and attending to any emergency.
All persons involved in the application of space spraying must wear overalls, protective gloves,
suitable respirator, ear plugs, goggles and boots.
Filter of the respirator must be periodically changed.
4.9.3 Pre space spraying activities
i) The steps listed below are to be followed in carrying out the space spraying of a designated area.
The maps of the area to be sprayed must be studied carefully before the spraying operation
begins.
The area covered should be at least 200 meters within the radius of the house where the dengue
cases are located.
Residents should be warned before the operation so that food is covered, fires extinguished and
pets are moved out together with the occupants.
The most essential information about the operation area is the wind direction. Spraying should
always be done from downwind to upwind, i.e. going against the direction of the wind.
ii) Information to be given to inhabitants
Time of spraying for example 08.00hours to 10.00hours

All doors and windows should be kept open
Dishes, food, fish tanks and bird cages should be covered.
Stay away from open doors and windows during spraying or temporarily leave the house and/or
the sprayed area until the spraying is completed.
Children or adults should not follow the spray squad from house to house.

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iii) To ensure proper quality of spraying the factors should be considered
1. Optimum spraying conditions

Spraying should be done in the early morning and late evening hours as adult aedes
mosquitoes are most active at these hours.
Spraying should not be done in the middle of the day, when the temperature is high as
convection currents from the ground will prevent concentration of the spray close to the
ground where adult mosquitoes are flying or resting thus rendering the spray ineffective.
Spraying should be carried out in steady winds (3 13 km/hr) while it should not be
carried out in strong wind conditions (> 13 km/hr).
In heavy rain, spraying should be stopped and the spray head of the ULV machine should
be turned down to prevent water from entering the blower.
Spraying is permissible during light showers as the mosquito activity increases with the
relative humidity.
2. Timing of application
Spraying should be carried out only when the right weather conditions are present and
usually only at the prescribed time. These conditions are summarized below.
Table 4.3 Time and favourable conditions for spraying
Most favourable conditions Average conditions
Time Early morning (0600* 1000 hrs) or Early to mid morning or late afternoon,
late evening (1600 1800 hrs) early evening
Wind Steady, between 3 13 Km/hr 0 3 km/hr
Rain No rain Light showers
Temperature Cool Mild
*For practical reasons, spraying should be commenced at 0800 hrs.
3. Frequency of Application
Spraying should be started in an area (residential houses, offices, factories, schools) as

soon as possible after a suspected DF/DHF cases from that area is reported.
At least two treatments should be carried out within each breeding cycle of the
mosquitoes (seven to ten days for Aedes). Therefore. Repeat spraying should be carried
out within seven to ten days after the first spraying.

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4.9.4 Types of Space spraying
i) Vehicle mounted space spraying
Doors and windows of houses and buildings in the area to be sprayed should be opened.
The vehicle is driven at a steady speed of 6 8 km/hr (3.5 4.5 miles/hr) along the streets. Spray
production should be turned off when the vehicle is not moving.
When possible, spraying should be carried out along streets that are at right angles to the wind
direction. Spraying should commence on the downwind side of the target area and progressively
move upwind.
In areas where the roads are narrow, and houses are close to the roadside, the spray head should
be pointed directly towards the back of the vehicle.
When there are inadequate roads to cover an area by the vehicle mounted fogging machine,
additional hand operated fogging machines need to be utilized to spray the inaccessible houses.
In dead end roads, the spraying should be done only when the vehicle is coming out of the dead
end, not while going in.
The spray head should be pointed at a 45 angle to the horizontal to achieve maximum throw of
droplets.
ii) Hand operated (Portable) thermal fogging
Thermal fogging with hand operated thermal foggers should be done from house to house, always
from downwind to upwind.
All windows and doors should be shut for half an hour after the fogging , to ensure good
penetration of the fog and maximum destruction of the target mosquitoes.
In single storey house, fogging can be done from the front door or through an open window
without entering every room of the house. All bedroom doors should be left open to allow
dispersal of the fog throughout the house.
In multi storey buildings, fogging should be carried out from upper floors to the ground floor and
from the back of the building to the front to ensure the good visibility of the operator along his
spraying path.
When fogging outdoors, it is important to direct the fog at all possible mosquito resting sites,
including hedges, covered drains, bushes and tree shaded areas.
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The most effective type of thermal fog for control is a medium / dry fog i.e. it should just
moisten the hand when the hand is passed quickly through the fog at a distance of about 2.5 3
meters in front of the fog tube. Adjust the fog setting so that oily deposits on the floor and
furniture are reduced.
iii) Back pack aerosol space spraying with ULV attachments
Stand 3 5 meters in front of the house and spray for 10 15 seconds directing the nozzle
towards all open doors, windows and eaves. If appropriate, turn away from the house and
standing in the same place, spray the surrounding vegetation for 10 to 15 seconds.
If it is not possible to stand three meters from the house due to the closeness of houses and lack of
space, the spray nozzle should be directed towards house openings, narrow spaces and upwards.
While walking from house to house, hold the nozzle upwards so that particles can drift through
the area. Do not point the nozzle towards the ground. In multi storey houses spraying is carried
out inside the houses.
Spray particles drift through the area and into houses to kill mosquitoes which become irritated
and fly into the particles. The settled deposits can be residual for several days to kill mosquitoes
resting inside houses and on vegetation not exposed to the rain.
This technique permits treatment of a house with an insecticide ranging from 1 to 25 grams in one
minute. The dosage depends on the discharge rate, concentration of insecticide applied, and time
it takes to spray the house.
4.9.5 General considerations
To obtain correct dosage, calibration of a machine should be done periodically, usually after 25 hours of
operation, or at any time when major maintenance is performed. Machines should be calibrated in a way
to ensure adherence of following parameters.
1. Optimum droplet size
Optimum droplet size should be 10 30 mm. Teflon coated slides should be used to measure the
droplet size of thermal fogging. Where water has been used to dilute the spray, water sensitive
papers stripes can be used to collect droplet for sizing. Treating the water sensitive paper with
ethyl acetate will make the stains more permanent.

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2. Flow rate
When using hand operated thermal fogging machine, at a walking speed of 60 meters per minute
and with track spacing of 10 meters, 600m can be sprayed in one minute. For an application rate
of 0.5 litre per hectare, the flow rate must be therefore be 30 ml/minute (500 ml 0.06).
Measurement of flow rate (ml/minute) can be calculated by either
i) Making a mark on the tank, then spray for one minute and measure the volume of liquid
needed to fill the tank back to the mark.
OR
ii) Adding a measured volume of an insecticide to the tank, then spray until the tank is
empty and timimg how long it takes to empty the liquid.
3. Flow rate of vehicle mounted thermal foggers
i) Outdoor applications
To calculate the output rate of vehicle mounted equipment, following formula can be used.
OUT PUT RATE (m2/minute) = vehicle speed (M/hour) x width of the track spacing (m)
10000 m2= hectare
If the insecticide label recommends an application rate of 0.5 litre of UL formulation per hectare, the flow
rate must be adjusted to deliver 0.5 litre per minute.
iii) For ULV fogging machine
Indoor space spray applications
Time required for space spraying a house can be calculated using the following formula.
Target application rate (ml/hectare) X area of the house (hectare)
Flow rate (ml/min)
4. Space Spray concentration
The WHO recommended targeted amount of active ingredient per unit area must remain within
the specified range given below. Susceptibility/resistance levels of the recommended insecticide target
species should be monitored regularly.

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Table 4.4 Insecticide suitable as cold aerosol sprays and for thermal fogs for mosquito control
Insecticide Chemical Dosage of ai (g/ha)

Cold Thermal
Malathion OP 112 693 500 600
Deltamethrin PY 0.5 1.0 -
Cypermethrin PY 13 -
Etofenprox PY 10 20 10 - 20
PY Synthetic pyrethroid, OP organophosphate

ai active ingredient
4.9.6 Evaluation of epidemic spraying
Epidemic spraying can be evaluated using the following indicators
i)Parous rate
A parous rate of 10% or less in comparison to a much higher rate before spraying indicates
the effectiveness of spraying.
However a low parous rate after spraying can occur in the absence of a marked reduction in
vector density. This can be attributed to the emergence of a new population of mosquitoes
which escaped the spray.
ii) Reduction in hospitalized cases
A reduction in hospitalized cases after the incubation period of the disease in humans (about
5-7 days) has elapsed indicates the effectiveness of spraying.

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4.10 Classification of Insecticides
Insecticides
Inorganic Organic
Plant Derivatives Petrolium Oils Synthetic Compounds
Chlorinated Hydrocarbones Organo-Phosphates Carbamates Pyrethroics
Eg : Eg : Eg : Eg
DDT Malathion Permethrin
Propoxur Deltamethrin
BHC Fenthion
Dieldrin Fenitrothion Cyfluthrin
Temephos
4.10.1 Insecticide formulations
Insecticide formulation = Active ingredient + Non-insecticidal ingredients
(Technical grade material) (Inert ingredients)
- enhance stability

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4.10.2 Commonly used formulations:
1. Technical grade
Active ingredient in its purest commercial form. Rarely used except in fogging and ULV
applications.
2. Dustable powder (DP) and Granule (GR)
Prepared by mixing the insecticide with an inert carrier. Used mainly to control lice and fleas.
3. Emulsifiable Concentrate (EC)
Active ingredient + solvent & emulsifier, diluted with water. May have a strong smell.
4. Emulsion oil-in-water (EW)
Active ingredient + solvent & combined surfactants. Diluted with water.
5. Capsule suspensions (CS)
Active ingredient encased in tiny plastic polymer capsule. Suspended in water.
6. Slow-release formulations
7. Solution
Active ingredient + solvent . Prepared as briquettes to provide controlled release of the insecticide.
8. Suspension concentrate (SC)
A solid active ingredient in a fluid . Diluted with water.
9. Wettable powder (WP) & Water dispersible powder (WDP)
Active ingredient + wetting agent & inert carrier. Suspended in water.

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Chapter 5
Identification of dengue vectors in Sri Lanka
5.1 Introduction
In Sri Lanka, Aedes aegypti and Ae. albopictus are important as vectors of dengue fever and dengue
haemorrhagic fever, and chikungunya. Correct identification of these species is important for dengue and
chikungunya control in the country.
Upto date, 48 Aedes species belonging to 11 subgenera were reported in Sri Lanka. The subgenera are
Aedimorphus, Cancraedes, Christophersiomyia, Diceromyia, Finlaya, Mucidus, Neomelaniconion,
Paraedes, Rhinoskusea, Stegomyia and Verrallina. Ae. aegypti and Ae. albopictus belong to subgeneus
Stegomyia (Amerasinghe, 1995, Kusumawathie 2008) .
In dengue vector surveillance, Aedes larval surveys are widely used to determine the vector density.
Therefore, this chapter mainly focus on Aedes larval identification. Since Aedes larvae share the habitats
with larvae of other genera , a generic key (Amerasinghe, 1995) for mosquito larvae was included in
order to differentiate Aedes larvae from larvae of other genera (annexure). In order to make the key user
friendly, illustrations were included in the key.
Scientific classification of Ae. aegypti and Ae. albopictus
Kingdom: Animalia
Phylum: Arthropoda
Class: Insecta
Order: Diptera
Family: Culicidae
Sub family: Culicinae
Genus : Aedes
Species: Ae. aegypti, Ae. albopictus

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5.2 External morphology of Aedes larva
The body of mosquito larva is divided into three major parts, viz, head, thorax and abdomen (Fig. 5.1).
Head
Thorax
Abdomen
Siphon
Anal segment
Fig 5.1 . External morphology of Aedes mosqioto larva
5.3 Identification characters of sub genus stegomyia (larvae)
Taxonomically important characters of larvae of sub genus Stegomyia of the genus Aedes
Larvae of the sub genus stegomyia can be differentiated from other sub genera of the genus Aedes by the
presence of following characters in Aedes (Stegomyia).
Head:
(1) Antennal shaft is smooth,

(2) 1-A is single,
(3) 4-C is with many branches,
(4) 5-C is single,
(5) 6-C is single or bifid,
(6) 7- C is with 2-3 branches and
(7) 6-C is situated in front of 5-C
Fig. 5.2 . Important cetae of the head of Aedes (Stegomyia) larva

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Abdomen:
(1) Seta 12- I is absent,
(2) number of comb scales < 15 and arranged in a single row (the comb scales may or may not attaches to
a chitinised plate on either side of the abdominal segment VIII) (Fig 5.3),
(3) Siphon is 1-3 times length of diameter at base and has no acus,
(4) Pecten teeth are about same size and arranged in a regular rank (Fig 5.4),
(5) Anal fan is with 4 pairs of setae (in some species, 4d-x is with 2 branches, fig 5.5),
(6) Chitinised saddle of segment X is usually rather small (Fig 5.5).
Fig 5.3 Abdominal segment VIII showing the arrangement of comb teeth

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Comb Scales
Fig. 5.3 Arrarrangement of comb teeth
Pecten
Fig. 5.4. Siphon of Aedes larva showing pectin

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4d-X
Fig. 5 5. Anal segment showing ventral brush (4d-X)
In Sri Lanka sub genus Stegomiya includes of 6 species, namely, Ae. aegypti, Ae. albopictus, Ae.
krombini, Ae. w-albus, Ae. novalbopictus and Ae. mediopunctatus. These species can be identified using
the following key.
1. Abdominal segment VIII with a semicircular chitinised plate to which the comb teeth are attached.
Comb is with 5 teeth and has no basal denticles
(Fig.5.6a). .Ae. mediopunctatus
Abdominal segment VIII without a semicircular chitnised plate (5.6b)..2
Fig 5.6a Fig 5.6b

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2.Comb teeth are with strong basal lateral denticles (5.7a).Ae. aegypti
Comb teeth are without strong basal lateral denticles, but finely fringed (5.7b)3
Fig 5.7a Fig 5.7b
3. Comb of 5 - 6 and pecten of 4 8 teeth (5.8a)Ae. w- albus
Comb of 8-12 and pecten of 7- 14 teeth (5.8b).4
Fig 5.8a Fig 5.8b

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4. Abdominal setae 1-VII is long and 2-3 branched.
4d-X is two branched (5.9a)..Ae. krombeini
Abdominal setae 1-VII is short and 4 branched. 4d-X is single (5.9b).5.
Fig 5.9a Fig 5.9b
5. Siphon is 3 times length of diameter at base. in most occasions,
saddle is complete. Abdominal setae 2-VII two branched (5.10a) Ae. novalbopictus
Siphon is 2 2 times length of diameter at base. in most occasions,
saddle is incomplete Abdominal setae 2-VII is single (5.10b)Ae. albopictus
Fig 5.10a Fig 5.10b

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During Aedes larval surveys, Ae. vittatus, Ae. macdougalli, Ae. pseudotaeniatus and Ae. crysolineatus are
most commonly encountered.
Ae. vittatus larvae can be differentiated from Aedes (Stegomyia).
In Ae. vittatus
(1) comb teeth are arranged in an irregular row and
(2) the antennal hair (1A) is branched.
Ae. macdougalli , Ae. crysolineatus and Ae. pseudotaeniatus have 25- 60 comb teeth arranged in a
triangular patch.

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5.4 Identification of adult Ae. aegypti and Ae. albopictus
Adult Ae. aegypti and Ae. albopictus can be identified by the arrangement of silvery bands of the dorsal
surface of the thorax. Ae. aegypti has lyre shaped markings (Fig 5.11a) while Ae. albopictus has a single
silvery line originating from the head and running through the middle of the thorax (Fig. 5.11b) .
Fig. 5.11a Fig 5.11b

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5.4 Key to the genera of mosquitoes of Sri Lanka (larva) (by Amerasinghe 1995)
1.Siphon absent. Seta 1 palmate on most abdominal terga (Fig 1a)Anopheles
Siphon present; seta 1 not palmate on abdominal terga (Fig 1b) 2
Fig. 1a Fig. 1b
2. Siphon short, attenuated, with saw toothed process near apex; adapted
for piercing plant tissues (Fig 2a) ...3
Siphon cylindrical in shape, if attenuated apically then without saw-toothed process (Fig 2b)..4
Fig 2a Fig 2b

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3.Antenna with part distal to setae 2,3-A as long as or longer than proximal part;
saddle with at most 2 weak setae ventrally (Fig 3a)...Coquillettidia crassipes
Antenna with part distal to setae 2,3-A less than 0.5 as long as proximal part;
saddle with 3-4 robust setae ventrally (Fig 3b)..Mansonia
Fig 3a Fig 3b
4.Ventral brush (4-X) with 1-2 pairs of setae (Fig 4a)...5
Ventral brush (4-X) with 4 or more pairs of fan like setae (Fig 4b)...7
Fig 4a Fig 4b

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5.Seta 5 or 6-P usually single, not stellate; 6-M 6-M or 7-T usually stout spine;
comb scales usually in single row (Fig 5a)....Tripteroides
Setae 5 and 6-P large; stellate; 6-M and 7-T never stout or spine-like; comb
scales usually in 2 or more rows (Fig 5b)6
Fig 5a Fig 5b
6.Abdominal segments IV-VI without stellate setae; maxillary palps well
developed or siphon index at least 6.0 (Fig 6a)..Topomyia
Abdominal segments IV-VI without stellate setae; maxillary palps
undeveloped; siphon index at most 4.0 (Fig 6b)..Malaya genurostris
Fig 6a Fig 6b

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7.Antenna enlarged, markedly curved and flattened; siphon with paired
hooks and branched setae at tip (Fig 7a).Aedeomyia catasticta
Antenna and siphon otherwise (Fig 7b)8
Fig 7a Fig 7b
8.Siphon with 3 or more pairs of setae 1-S (Fig 8a).Culex
Siphon with 1 pair of setae 1-S (Fig 8b)...9
Fig 8a Fig 8b

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9.Siphon without pecten (Fig 9a)...10
Siphon with pectin (Fig 9b)..13
Fig 9a Fig 9b
10.Lateral palatal brush with 6-10 curved stout rods; comb scales absent
(Fig 10a)....Toxorhynchites
Lateral palatal brush with numerous fine simple or pectinate filaments;
comb scales present (Fig 10b)...11
Fig 10a Fig 10b

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11.Distal part of antenna with joint, apical part freely movable;
setae 2,3-A attached at joint (Fig 11a)...Mimomyia sub genus Etotleptiomyia
Distal part of antenna without joint; setae 2,3-A attached apically or
sub apically (Fig 11b)..12
Fig 11a Fig 11b
12.Abdominal segment VIII with a dorsal sclerotized plate, sometimes also
present on segment VII; seta 1-A large, with 4 or more branches (Fig 12a)..Orthopodomyia
Abdominal segments VII and VIII without sclerotized plates;
seta 1-A minute, usually single or bifid (Fig 12b).Armigeres
Fig 12a Fig 12b

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13.Seta 1-S attached near siphon base (Fig 13a)....14
Seta 1-S attached beyond basal 0.33 of siphon (Fig 13b). ..15
Fig 13a Fig 13b
14.Seta I C simple; pectin with at least 3 teeth, usually more (Fig 14a)....Hodgesia
Seta 1 C strongly barbed; pectin with at most 2 teeth (Fig 14b)..Ficalbia minima
Fig 14a Fig 14b

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15.Distal part of antenna with joint, apical part freely movable; setae 2,3-A
attached at joint (Fig 15a)...Mimomyia sub genus Mimomyia
Distal part of antenna without joint; setae 2,3-A attached apically or
sub apically (Fig 15b).16
Fig 15a Fig 15b
16.Venter of head with hypostomal suture absent or incomplete, not reaching the posterior tentorial pit;
comb scales usually attached to comb plate; seta 5 and / or 6-C often spine-like
(Fig 16a).Uranotaenia
Venter of head with well developed hypostomal suture extending from level of mentum to posterior
tentorial pit; comb scales not borne on comb plate; setae 5,6-C not spine like
(Fig 16b). ..17
Fig 16a Fig 16b

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17. Seta 4-C large, multibranched, subequal in size to 7-C; 6-C with two
two unequal branches, rarely single (Fig 17a)..Heizmannia
Seta 4-C small, variously branches, less than 0.5 length of 7-C; if 4c longer,
6C never with two unequal branches or head sub quadrate (Mucidus)
or seta 4 C closer to 6C than to 5C (Stegomyia) (Fig 17b)...Aedes
Fig 17a Fig 17b

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5.5 Key to the genera of mosquitoes of Sri Lanka (Adult) (by Amerasinghe 1995)
1.Proboscis long, recurved, posterior margin of wing emarginated just
beyond tip of vein CuA Toxorhynchites
Proboscis straight or only slightly curved, posterior margin of wing
evenly rounded or only slightly emarginated ..2
2.Scutellum evenly rounded, with evenly distributed setae; female
maxillary palp almost as long as proboscis, male palp long, apex expanded
to form a club, abdominal segments with only occasional scales .Anopheles
Scutellum trilobed, with setae in 3 groups; female maxillary palp markedly
shorter than proboscis, male palp variable in length, not clubbed at apex
(except in some Mimomyia) abdominal segments densely covered by scales ...3
3.Mid and hind femora with large sub erect apical scale tufts; female antennal flagellomeres
short and thick, basal flagellomere with prominent scale tuft; male antenna with the two terminal
flagellomeres markedly thickened Aedeomyia catasticta
Femora and antenna otherwise .4
4.Fore and mid tarsomere 1 distinctly longer than other 4 tarsomeres
combined, tarsomere 1 of fore and mid legs short, about as long as wide;
wing spotted, as in many species of Anopheles...Orthopodemyia
Fore and mid tarsomere 1 shorter than other four tarsomeres combined,
tarsomere 4 of fore and mid legs much longer than wide; wing variable ...5
5.Prespiracular area with setae or covered with scales ..6
Prespiracular area bare ....9

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6.Scutum with conspicuous median longitudinal double line of broad
scales. Usually white or silver; other scutal scales narrow ..7
Scutum without median longitudinal double line of broad pale or dark
scales; broad or narrow pale scales may be present along scutal margin .....8
7.Proboscis swollen apically, upturned and with long apical setae ..Malaya genurostris
Proboscis slender, if at all only with slightly swollen labellum at tip,
not upturned or with long apical setae ......Topomyia
8.Cell R2 of wing always shorter than vein R2+3; vein 1A reaching wing
margin before, or at most very slightly beyond base of vein mcu ....Uranotaenia
Cell R2 at least as long as vein R2+3; vein 1A reaching wing margin well
beyond base of vein mcu Tripteroides
9.Meso postnotum with setae; scutum with broad metallic decumbent scales .Heizmannia
Meso postnotum without setae; scutum with narrow scales ..10
10.Outstanding scales on outer half of wing with emarginated tips;
vein 1A reaching wing margin before, or at most, very slightly beyond
base of vein mcu Hodgesia
Outstanding scales on outer half of wing without emarginated tips;
vein 1A reaching wing margin well beyond base of vein mcu, except
Aedes subgenus Cancraedes ..11
11.Post spiracular setae present (note presence of alveoli if setae missing) 12
Post spiracular setae absent or obscured by heavy scalation .....14

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12.Dorsal surface of wing with broad, asymmetrical dark and pale scales
Intermixed ...Mansonia
Dorsal surface of wing with narrow scales, if broad, then not asymmetrical .13
13.Proboscis rather stout, laterally compressed and curved downwards; occiput with broad
decumbent scales ..Armigeres subgenus armigeres
Proboscis more slender, not laterally compressed or notably curved;
occiput with at least some decumbent scales narrow(except in subgenus
Stegomyia) ..(in part) Aedes
14. Alula bare or with broad flat decumbent scales ...Mimomyia
Alula fringed with narrow scales or moderately broad erect scales ....15
15.Postspiracular area covered with broad scales .16
Post spiracular area without scales ...17
16.Post spiracular area dark scaled dorsally and white scaled ventrally;
female palpus 0.4-0.7 length of proboscis ...Armigeres subgenus Leicesteria
Post spiracular area with pale scales only; female palpus less than 0.4 length of
Proboscis .(in part) Aedes
17.Female antennae with flagellomere 1 approx. three times the length
of flagellomere 2; male proboscis greatly swollen on distal third or more;
palpus very short, seldom longer than clypeus ..Ficalbia punima
Female antenna with flagellomere 1 approx. equal length of flagellomere 2; male proboscis
only slightly swollen apically; palpus longer than clypeus .18

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18.Pulvilli absent; tarsal claws prominent in all legs; abdominal terga with
purple metallic scales; wing with yellow scales ...Coquillettidia
Pulvili well developed at least on hind leg; tarsal claws usually small,
inconspicuous, abdominal terga and wing scales otherwise .Culex

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5.6 Charcaters of the subgenus Stegomyia of the genus Aedes
a. Segment VIII is broader and not completely retractile.

b. Cerci shorter and broader.
c. Tarsal claws all simple or toothed, at least on fore and mid legs.
d. If tarsal claws simple, these are black species with snow white markings and white tarsal bands
on tarsi.
e. If tarsal claws toothed, sternite VIII is smaller and less prominent, dorsal surface of head with
many flat scales.
f. Scutellar scales broad and flat, at least on mid lobe; species with conspicupous ornamentation.
g. Scutellar scales broad and flat on all lobes.
h. Proboscis dark.
i. Apn lobes with broad flat scales.
Key to species of the subgenus Stegomyia of the genus Aedes
1.Mesonotum marked with a pair of lateral curved white lines, and
usually also with a pair of submedian yellowish lines; two dots of white
scales on clypeus (Tibiae without white rings). ....aegypti
Mesonotum otherwise marked. Clypeus without white scales. Palpi with
white scaling; proboscis normally thick and about length of fore femur
(Tibiae without white rings) ..2
2.Mesonotum with a broad white anterior stripe, or with a white patch or
pair of patches in front..3
Mesonotum with a narrow median white line running nearly the whole length ..4
3.Midfemur with a median white spot on anterior surface; mesonotum with
several white patches..w-albus

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Midfemur without a white spot. Mesonotum with a broad median white stripe,
narrowing posteriorly.....mediopunctatus
4.Midfemur without a white spot. White scales on pleurae arranged in irregular patches.
A patch of white scales in front of wing-root only.
Pale markings on mesonotum all silvery white. Abdomen with silvery basal bands on
dorsum.albopictus, novalbopictus and krombeini

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5.7 Identification guide for the sub genera of Aedes : larvae (based on so far described species)
1.Mouth brushes adapted for predacious habits. Both Osc and Isc hairs single.
Anal fan of close set branched hairs extending along whole length of anal
segment ventrally .Mucidus
Mouth brushes not adapted for predacious habits. Isc usually of 2-10 branches,
but, if both pairs are single anal fan does not extend along whole of ventral
surface of segment ....2
2.Comb of 5-19 large teeth in a single row or of 6-14 teeth in an irregular row
or more or less in 2 rows ..3
Comb of 20-70 teeth, usually small arranged in several rows or more or patch ...11
3.Antennal shaft with small specules or spines. Basal pectin teeth with lateral
denticles and smaller or at least not longer than more distal teeth ..4
Antennal shaft smooth .....11
4.The 2-3 pecten teeth furthest from base of siphon more widely spaced than
those nearer base, and usually distinctly large than basal teeth ..5
All pectin teeth evenly spaced and all about same size, those furthest from
base being but little larger than remainder ..8
5.Frontal hairs B and C both with several branches ...6
Frontal hair B single (unbranched). Frontal hair C, 1-3 branches. Siphon
2 -3 times length of diameter at base .Aedimorphus (part)

130
6.All pectin teeth with lateral denticles except most distal tooth in some
Individuals ..7
The most distal 2-3 pecten teeth without lateral denticles ....Aedimorphod (part)
7. Antennal shaft with few minute specules and tuft of 3 fairly stout branches,
nearer base than apex. Comb usually of 10 teeth. Pectin teeth with long
sharp point. Frontal hairs B and C each usually 3 branched ..Verallina
Antennal shaft with fairly numerous strong spicules and with tuft of
stout 5 branches at middle. Comb usually of 6-8 teeth. Pecten teeth smaller,
frontal hairs B and C each with usually 5 or more branches Neomelaniconion linetopennis
8.Frontal hairs A,B,C and d all well developed, and each with a number of fairly
long branches ...Finlaya
Hair d much smaller than other 3 pairs and with few short fine branches.
Comb about 14 teeth in a single row. Isc , 2 branched. Pectin of about 16
comparatively small short teeth .Chrystopersiomyia
9.The most distal pectin tooth widely spaced from main rank and attached
between hair tuft and apex of siphon ..Aedimorphos
Comb in a single regular row. Pecten teeth (< 14) all in one rank, non
widely spaced. Frontal hair C single, B single or bifid ..10
10.Pecten teeth with lateral denticles along one side from base to apex ..Diceromyia
Pectin teeth with basal lateral denticles only Isc with at most 3 branches
usually single ..Stegomyia

131
11.One or 2 pairs of frontal hairs single and very long, longer than whole head Finlaya
None of the frontal hairs longer than the whole head ....12
12. Several of the more distal pectin teeth bare, without lateral denticles or
fringe and usually larger than the more basally situated teeth .......13
All pectin teeth with lateral denticles or fringed, and usually all about same size .14
13.Pecten with 2-3 large simple distal teeth markedly detached from the more
basal teeth. Pectin confined to about basal of tube ..Aedimorphos
Pectin either without markedly detached distal teeth or if with such teeth
the pectin extends for much more than basal of siphon ..............Finlaya
14.Siphonal tuft very near apex of siphon and of 3 long branches.
Antennal tuft of 3-4 branches, nearer base than apex ....Rinoskusia
Siphonal tuft not usually more than 2/3 of length from base of tube.
Antennal tuft usually at about middle of shaft or nearer apex than this ..Finlaya
(Frontal hair more forward arranged in a straight line. 4,5,6 in front of 7C. Antennal hair
Single macdougalli)

132
Reference :
Aitken T.H.G., Downs W.G., Shope R.E., 1977. Aedes aegypti strain fitness for yellow fever
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1

1.1 Origin and Natural history

Medical Research Institute and Dengue coordination Unit - 2011

Medical Research Institute and Dengue coordination Unit - 2011

Fig. 1.1 Countries/Areas at risk of dengue transmission (WHO 2008)

Medical Research Institute and Dengue coordination Unit - 2011

1.2 Dengue situation in Sri Lanka

Medical Research Institute and Dengue coordination Unit - 2011

Dengue cases and deaths in Sri lnka from 2000 - 2010

Provincial distribution of Dengue cases in Sri Lanka 2010

Medical Research Institute and Dengue coordination Unit - 2011

1.3 Seasonal Distribution

1.4 The virus

1.5 The host

1.6 Transmission of the dengue virus

Fig 1.6 Transmission of Dengue virus by Aedes aegypti

Medical Research Institute and Dengue coordination Unit - 2011

Altitude is an important factor in limiting the distribution of Ae .aegypti. In India Ae.

IV) Transport of mosquito vector

Ground transportation also provides an efficient mechanism of dissemination of both

V) Threshold mosquito density for dengue transmission

Medical Research Institute and Dengue coordination Unit - 2011

VI) Flight range.

VII) Feeding behaviour

VIII) Extrinsic incubation period

Medical Research Institute and Dengue coordination Unit - 2011

IX) Daily survival rate, longevity and gonotrophic cycle

Most, if not all investigators working on mathematical models of arthropod borne

Medical Research Institute and Dengue coordination Unit - 2011

Yellow fever in Africa & tropical areas of America

i) Afrotropical Region (Subsaharan Africa) Selected reference

Aedes (Stegomyia) aegypti (Linnaeus) Trpis & Hausermann (1986)

Aedes (Stegomyia) albopictus (Skuse) Rodhain & Rosen (1997)

Aedes (Stegomyia) luteocephalus Rodhain & Rosen (1997)

Aedes (Diceromyia) furcifer Edwards Rodhain & Rosen (1997)

Aedes (Diceromyia) taylori Edwards Rodhain & Rosen (1997)

Medical Research Institute and Dengue coordination Unit - 2011

ii) South Pacific Islands and Australian Region Selected reference

Aedes (Stegomyia) cooki Belkin Rodhain & Rosen (1997)

Aedes (Stegomyia) polynesiensis Marks Rodhain & Rosen (1997)

Aedes (Stegomyia) rotumae Belkin Rodhain & Rosen (1997)

Aedes (Stegomyia) scutellaris (Walker) Rodhain & Rosen (1997)

Ochlerotatus (Finlaya) notoscriptus (Skuse) Rodhain & Rosen (1997)

iii) Oriental region including South East Asia

Aedes (Stegomyia) aegypti (Linnaeus) Hammon et al(1960), Huang(1961)

Rodhain & Rosen (1997)

Huang (1979), Rodhain & Rosen (1997)

Ochlerotatus (Finlaya)niveus sub group Rudric (1986)

iv) Americas (including the Carribean Islands)

Aedes (Stegomyia) aegypti (Linnaeus) Rodhain & Rosen (1997)

Aedes (Stegomyia) albopictus (Skuse) Rodhain & Rosen (1997)

Medical Research Institute and Dengue coordination Unit - 2011

Aedes aegypti Aedes albopictus

Fig. 2.1 Adults of Ae. Aegypti and Ae. albopictus

2.2 Life cycle

Fig 2.2 Life cycle of Aedes mosquito

Medical Research Institute and Dengue coordination Unit - 2011

Egg shell has a mosaic pattern

Fig 2.3 Eggs of Aedes

Medical Research Institute and Dengue coordination Unit - 2011

Fig 2.6 Pupa

Medical Research Institute and Dengue coordination Unit - 2011

Fig 2.7 Aedes aegypti Aedes albopictus