Journal of Mosquito Research 2015, Vol.5, No.14, 1-8
3
Sampling, Rearing and Identification of adult
Anopheles
mosquitoes
Three villages (approximately 20 Km apart) were
selected and sampled from each study site. That is,
Mbuinjeru, Ndindiruku and Murinduko villages in
Mwea scheme; Kamagaga, Kobura and Wagai villages
in Ahero scheme. Indoor sampling was carried out as
described by Mathenge et al., (2004); Kamau et al.,
(2007). Briefly, 15 houses were randomly sampled for
gravid and blood fed female
Anopheles
mosquitoes
and a total of 200 mosquitoes from each village were
collected. This was conducted between 0600Hrs and
0800Hrs. Outdoor sampling was done overnight using
CDC light traps model 512 (John W. Hock Company,
Gainesville, Fl, USA) with each trap placed a meter
from respective households after which sorting was
done for gravid and blood fed
Anopheles
mosquitoes.
The mosquitoes were morphological classified in
situ
into
An. gambiae
s.l
or
An. funestus
complex by
taxonomic methods of
and kept separately in paper cups. The mosquitoes
were maintained live and subsequently transferred to
insectary where standard procedures for rearing
Anopheles
mosquitoes were followed. Individual
females were subsequently sorted based on their
physiological status. Individual gravid females were
placed in tubes and allowed to oviposit and F
1
families
raised separately. After emergency, adult mosquitoes
were provided with 10% sugar solution soaked in
cotton wool for 2-3 days before conducting bioassays.
In cases where F
1
was less than 100, that is, in
Kamagaga and Wagai for
An. arabiensis
and Kobura
for
An. funestus
, F
2
was used for the bioassays. After
oviposition, the field collected mosquitoes were used
to determine which member of the species complex
they belong to, thus their offspring. DNA was
extracted by alcohol precipitation method of Collins et
al.,
(
1987) and the species-specific analysis done
using PCR method of Scott et al., (1993).
Insecticide resistance testing
Bioassays were conducted using 2-3 day old
mosquitoes for insecticides in each of the four classes
of insecticides recommended by WHO; pyrethroids
(permethrin and deltamethrin), organochlorides (DDT),
carbamates (bendiocarb) and organophosphates
(fenitrothion). Assessment was done using papers
impregnated with 0.75 permethrin, 0.05 deltamethrin,
0.04 DDT, 0.01bendiocarb or 1.00% fenitrothion
insecticides following WHO guidelines (WHO, 2005).
Briefly, 20–25 mosquitoes (four replicates each) were
exposed to insecticide-treated papers for an hour
(permethrin, bendiocarb or DDT) or two hours
(fenitrothion).
An. gambiae
Kisumu susceptible strain
(KSM Strain) was used as positive control while
negative control comprised the F
1
mosquitoes exposed
to paper treated only with silicon oil solvent.
Knock-down (KD) counts were recorded in every 10
minutes. After the exposure period, the mosquitoes
were transferred into individual holding tubes without
insecticide treated papers and provided with 10%
sugar solution and mortality recorded 24 hours later.
Determination of Blood meal sources and
Plasmodium falciparum
infection
Mosquito abdomens with visible blood meals were
individually tested for the source of the blood
according to Beier et al. (1988). A total of 405 female
An. arabiensi
s from Mwea study site were assayed. In
Ahero, a total of 169
An. arabiensis
and 269
An.
funestus
abdomens were assayed for the sources of the
blood meal. A total of 1200 heads and thoraces of all
the collected mosquitoes from both study sites (after
oviposition and stunning at 4˚C and abdomen removal)
were tested for the presence of
P. falciparum
by
sandwich Enzyme Linked Immunosorbent Assay
(ELISA) method of Wirtz et al., (1987).
Results
A total of 600 female
Anopheles
gambiae
s.l
mosquitoes were collected (355 indoor and 245
outdoor) from Mwea sentinel site as shown in Table 1.
All the
Anopheles gambiae
s.l was identified as
An.
arabiensis
by PCR (Table 2). In Ahero,
An. gambiae
s.l
was 250 (115 indoor and 135 outdoor) and
An. funestus
was 350 (195 indoor and 155 outdoor) as shown in
Table 1. All the
An. gambiae
s.l were identified as
An.
arabiensis
while
An. funestus
members were
Anopheles
funestus sensu stricto
Giles, 1900 (342)
, Anopheles
rivulorum
Leesoni, 1935 (4)
, Anopheles leesoni
Evans,
1931 (2) and
Anopheles parensis
Gillies, 1935 (2) as
shown in Table 2.
A total of 100 (four replicates of 25), 2-3 days old
females of each identified
Anopheles species
were
assayed against each insecticide as shown in Tables 3.
In Mwea, mortality 24h post-exposure was below the
