Anopheline mosquitoes behaviour and entomological monitoring in Southwestern Ethiopia

Abstract

Background & objectives: Despite a tremendous expansion in the financing and coverage of malaria control programmes, the disease continues to be a global health threat. This study was conducted to assess the entomological parameters of anopheline mosquitoes, viz. species composition, abundance, longevity, behaviour and infectivity rates in Kersa district, Jimma zone, southwestern Ethiopia.
Methods: Mosquito collection was carried out from each selected household in each of the nine selected study villages of Kersa district, using CDC light-traps and pyrethrum spray catches (PSCs) for seven months (June to December 2014). Mosquito count data were log transformed before analysis and the data were analyzed using SPSS software package version 16.0. Analysis of variance (ANOVA) was employed to compare means and Tukey’s post-hoc test was used for mean separation.
Results: In total, 1559 adult female anopheline mosquitoes, representing at least three species were collected from the study villages. Of these, 1122 were collected by CDC light-traps and the rest 437 were collected by PSCs. Anopheles gambiae s.l. (71.8%) was the most abundant species, followed by An. coustani s.l. (22%) and An. pharoensis (6.2%). The mean monthly density of anopheline mosquito species was highly significant (p < 0.001). Significantly (p

Full text

INTRODUCTION
Despite a tremendous expansion in the nancing and
coverage of malaria control programmes that has led to a
wide-scale reduction in malaria incidence and mortality,
the disease continues to be a global health threat; and has
resulted in 429,000 deaths from the estimated 212 mil-
lion cases in the year 2015, alone1. Furthermore, only 59
out of 103 countries with ongoing malaria transmission
are on the track of meeting the Millennium Development
Goal (MDG) target of reversing the incidence of malaria;
among them 52 were on the track of reducing malaria case
incidence rates by 75% till 20152. Considering substantial
reduction of global funds for malaria control and elimi-
nation in recent years and the overwhelming growing
evidence of antimalarial drug resistance combined with
insecticide resistance of vectors3, there is no assurance
that the recent gains achieved in disease control cannot be
thrown away cheaply.
The disease remains a long-standing problem in
Ethiopia, where all the four human malaria parasites are
prevalent in the country, with Plasmodium falciparum
being the most widely distributed and dominant parasite
species, followed by P. vivax, P. malariae and P. ovale4. In
Ethiopia, approximately 75% of the total area is estimated
to be malarious, with 68% of the total population being
at risk of infection5. Malaria transmission peaks twice in
a year, from September to December and April to May,
and the transmission is unstable where major epidemics
occurs due to climatological anomalies5. Anopheles ara-
biensis, member of An. gambiae complex, An. funestus
group, An. pharoensis and An. nili, are widely distributed
Anopheline mosquitoes behaviour and entomological monitoring in south-
western Ethiopia
Kidane Lelisa1, Abebe Asale2, Behailu Taye3, Daniel Emana4 & Delenasaw Yewhalaw4-5
1Department of Biology, Dilla University, Dilla; 2Department of Biology, Jimma University, Jimma; 3Department of Wildlife and Ecotourism
Management, Gambella University, Gambella; 4Department of Medical Laboratory Sciences and Pathology; 5Tropical and Infectious Diseases
Research Center, Jimma University, Jimma, Ethiopia
ABSTRACT
Background & objectives: Despite a tremendous expansion in the nancing and coverage of malaria control pro-
grammes, the disease continues to be a global health threat. This study was conducted to assess the entomological
parameters of anopheline mosquitoes, viz. species composition, abundance, longevity, behaviour and infectivity
rates in Kersa district, Jimma zone, southwestern Ethiopia.
Methods: Mosquito collection was carried out from each selected household in each of the nine selected study villages
of Kersa district, using CDC light-traps and pyrethrum spray catches (PSCs) for seven months (June to December
2014). Mosquito count data were log transformed before analysis and the data were analyzed using SPSS software
package version 16.0. Analysis of variance (ANOVA) was employed to compare means and Tukey’s post-hoc test
was used for mean separation.
Results: In total, 1559 adult female anopheline mosquitoes, representing at least three species were collected from the
study villages. Of these, 1122 were collected by CDC light-traps and the rest 437 were collected by PSCs. Anoph-
eles gambiae s.l. (71.8%) was the most abundant species, followed by An. coustani s.l. (22%) and An. pharoensis
(6.2%). The mean monthly density of anopheline mosquito species was highly signicant (p < 0.001). Signicantly
(p <0.05) higher population of An. gambiae s.l. were trapped indoor than outdoor. However, outdoor mean densities
of An. pharoensis and An. coustani s.l. were signicantly (p < 0.001) higher than indoor mean densities. The longev-
ity of An. gambiae s.l. was higher in the months of June, July and August (mean 7.32 days) and lower in the months
of October, November and December (mean 2.94 days). Two An. gambiae s.l. specimens were found positive for
Plasmodium vivax 210 polymorphs and the overall infectivity rate was estimated to be 1.04%.
Interpretation & conclusion: This study could contribute to the understanding of anopheline mosquitoes with respect
to their composition, dynamics, distribution and behaviour in Kersa district, for evidence based malaria vector control
programmes, mainly in the appropriate timing of the indoor residual spray programme.
Key words Anopheles; Ethiopia; infectivity rate; malaria; mosquitoes; parity rate
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241
anopheline vector species in the country, with former be-
ing the primary vector 6-10.
Disease control strategy in Ethiopia includes advoca-
cy, community mobilization, and communication activi-
ties for behaviour change in all populations to household
level, creating demand for interventions and utilization of
services through health extension workers (HEWs)11-13.
Furthermore, the Government of Ethiopia has set an am-
bitious national goal in 2005 to provide 100% coverage
of insecticide treated nets (ITNs) in malarious areas, with
a mean of two ITNs per household; to scale-up indoor
residual spraying (IRS) in households with insecticide, to
cover 30% of households targeted for IRS; and scale-up
the provision of case management with rapid diagnostic
tests (RDTs) and artemisinin-based combination thera-
pies (ACTs)14-17.
However, decreased sensitivity of parasites to
drugs18-19, decreased susceptibility of vector(s) to pub-
lic health insecticide(s)20-27, the behavioural plasticity
of vector mosquitoes following the application of long-
lasting insecticidal nets (LLINs) and IRS28-31 and residual
transmission that is acquired mainly through, out-door
or early biting32 could jeopardize the envisaged disease
control and elimination programme. In addition to this,
better understanding of local malaria vector behaviour,
their ecology and microclimate would permit a better un-
derstanding of malaria transmission in one particular set
up in order to optimize control strategies aimed at reduc-
ing man-vector contact. Such critical information can be
obtained through longitudinal monitoring and evaluation
of vector population before and after the application of
control measures such as LLIN and IRS. Therefore, in this
study an entomological assessment regarding anopheline
mosquito composition, abundance, parity rate, longevity,
resting behaviour, infectivity rates and other entomologi-
cal parameters were carried out pre- and post-IRS opera-
tion in Kersa district, southwestern Ethiopia.
MATERIAL & METHODS
Study area and period
The study was conducted from June to December
2014 in Kersa district, southwestern Ethiopia. It is located
in the southwestern part of Jimma zone, Oromia region-
al state, 333 km southwest of Addis Ababa. The study
area lies between latitudes 7°35ꞌ–8°00ꞌ N and longitudes
36°46ꞌ–37°14ꞌ E. The altitude ranges between 1740 and
2660 m above sea level. The district is malarious; the eco-
logical condition in the district favours the existence of
Anopheles mosquitoes responsible for malaria transmis-
sion. Malaria is the most prevalent seasonal disease in the
area accounting for 77.1% of all the reported diseases in
the health center during 2006 and 2007. The district has
more or less homogenous characteristics of house style
with walls made of wood and mud plastered and roofs
covered with grass. The community practice similar per-
sistent agricultural socioeconomic activity, and majority
of them are Oromo ethnic33. Out of 32 “Kebeles” (smallest
administrative unit in Ethiopia) that existed in the district,
20 of them are categorized as highly malarious, 10 as mild
and two as malaria free district (District Health Oce).
Study design
A longitudinal study was conducted using entomo-
logical parameters such as Anopheles species composi-
tion, density, vector population dynamics, behaviour,
longevity and vector infectivity in nine villages selected
from three sentinel sites in Kersa district of Jimma zone,
southwestern Ethiopia. In Ethiopia, there is routine an-
nual national spray programme and distribution of LLINs
as part of vector control interventions, with replacement
of LLIN being done every three years. Intervention cam-
paign usually start in the second half of August and last for
1 to 2 months. At this particular study site, bendiocarb (0.4
g/m2) has been used for indoor residual spray and LLINs
(PermaNet 2.0) have been distributed since September
2013. The dynamics of vectors was monitored from the
month of June to December 2014.
Monthly mosquito sampling and identication
Three “Kebeles” namely Bulbul, Gelo and Ankaso
were selected randomly from 20 highly malarious “Ke-
beles”. A total of nine villages (three from each “Ke-
bele”) were used for entomological assessment. A total
nine houses were randomly selected (one per village) for
whole night light-trap collections (LTCs) using the Cen-
ter for Disease Control and Prevention (CDC) light-traps
(Model 512; John W. Hock Co., Gainesville, FL, U.S.A.).
Adult female Anopheles mosquito collections were car-
ried out indoor and outdoor in each of the selected houses
twice a month from June to December 2014. The CDC
light-trap was set to run between 1800 to 0600 hrs and
was set inside the bed room (untreated bednet were also
provided to the inhabitants), whereas outdoor collection
was set in the radius of 15–20 m surrounding the indoor
collected houses or nearby cattlesheds.
Pyrethrum spray catch (PSC) was employed to collect
indoor resting Anopheles mosquitoes from 0600–0730
hrs in 60 houses, 20 from each of three villages namely,
Sarado, Waddeyi and Warsu which were dierent from
those used for LTCs. Anopheline mosquitoes were sam-
pled from each house once a month, from June to Decem-
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J Vector Borne Dis 54, September 2017
242
ber 2014. Prior to PSC, the inhabitants were instructed to
empty the house, any openings that could allow mosquito
escaping were closed and entire oor was covered with
a white sheet of cloth made of cotton. Then a protected
sprayer (person) sprayed the room with Mobil it (Bioy-
gon SC, Jonhanson and Sun. Inc. USA) for about 5 min
and left closed for 15 min. Subsequently, the sheet was
brought outside the room and knockdown mosquitoes
were inspected and non-anopheline mosquitoes such as
Culex were identied visually using key characters such
as wing and abdominal appearance. The abdominal status
of all collected mosquitoes were observed with exception
of Culex samples and sent to Asendabo Field Vector Biol-
ogy of Jimma University, Jimma for further morphologi-
cal identication using standard keys34-35.
Abdominal status detection
Following the guidelines of WHO36 the blood meal
digestion stages of female Anopheles collected by LTCs
and PSCs were identied by the help of hand lens and
then grouped as unfed, freshly fed, half-gravid, and grav-
id. The unfed and freshly fed groups were used for ovary
dissection, for determining the parity rate and longevity
of mosquitoes. Fed and gravid specimens of An. gambiae
s.l., An. pharoensis and An. coustani were preserved on
silica gel. Head and thorax region of unfed and parous An.
gambiae s.l. were also preserved on silica gel for circum-
sporozoite protein (CSP) detection along with the fed and
gravid specimen of this species36.
Determination of parity
Ovaries of unfed anopheline mosquitoes collected by
LTCs method were dissected36, following specimen iden-
tication at species level, and abdominal status detection
after immobilization using chloroform36. Briey, the legs
and wings of a mosquito were removed, and then placed
on a slide in a drop of phosphate buer saline solution
(PBS). While holding one dissecting needle on the thorax,
under a dissecting microscope, the ovaries were removed
by breaking the abdominal wall in the region of the 7th
to 8th sclerite, and then by pulling the tip of the abdomen
away from the rest of the body with a second needle held in
the right hand. Then the two lobed ovaries were separated
and allowed to air dry and liquid if any was absorbed using
lter paper. The ovaries, were examined for ovary trache-
oles under compound microscope using the 10x objective,
and when necessary, a conrmation was made using 40x
objective. The ovaries in which the terminal skeins of the
tracheoles had become uncoiled were considered to be
parous, while the ovaries with coiled skeins were consid-
ered to be nulliparous36.
Determination of infectivity
Head-thorax of unfed parous, fed, half-gravid and
gravid specimens of An. gambiae s.l. collected by LTCs
and PSCs was used for CSP detection.
The head-thorax of a mosquito was removed with a
sharp clean surgical blade on lter paper and then trans-
ferred to labeled eppendorf tube of 1.5 ml by clean forci-
pes; while the rest parts of the mosquito were preserved
for further study. Then 50 µl of blocking buer (BB)
containing IGEPAL CA-630 (Sigma-Aldrich, USA) was
added to each of the sampled head-thorax; grinded and
homogenized very well through vortexes, handshaking
and using new non-absorbent plastic pestle. About 200
µl BB was added to each labeled eppendorf tube and kept
in –20°C.
About 50 µl of diluted capture monoclonal antibod-
ies (MAb) (CDC, Atlanta, USA) was added to ELISA
plates, and incubated for 1 h at room temperature. Then
the capture MAb was aspirated by multichannel pipettes
and completely drained by banging plates sharply on an
absorbent tissue paper to ensure complete dryness. Fol-
lowing this, each well was completely lled with BB and
incubated for 1 h at room temperature. The plates with BB
were then aspirated using multichannel pipettes and the
remaining buer droplets were drained again by banging
plates sharply on an absorbent tissue paper. About 50 µl
of positive control (recombinant protein antigen, CDC,
Atlanta, USA) and 50 µl (per well) of the negative controls
(homogenate of laboratory reared female An. gambiae
s.l.) were added in the rst two column of each plate. Then,
50 µl of each mosquito homogenate was loaded to the
remaining wells of the plate, with careful crosschecking
of the correct correspondence of codes between the plate
wells and labels on each eppendorf tube containing the
homogenate. Then the plate was covered with aluminum
foil and incubated for 2 h at room temperature in subdued
light. The homogenate was then removed and the wells
were washed twice with 150 µl PBS-Tween 20 through
lling and emptying. Then 50 µl Peroxidase-conjugated
MAb was added and incubated for 1 h at room tempera-
ture. The enzyme conjugate from the wells were aspi-
rated and washed three times with PBS-Tween 20. Then
about 100 μl of 2,2ꞌ-Azino-bis(3-ethylbenzthiazoline-6-
sulfonic acid) (ABTS) (Sigma-Aldrich, USA) substrate
per well was added to each well and incubated for 30–60
min. Appearance of dark green colour marked positive
reaction as described in a study by Wirtz et al37. Lastly,
the results of each tested mosquito sample were recorded.
The sporozoite rates were calculated as the proportion of
mosquitoes containing malaria sporozoite antigen to the
total samples tested by ELISA.
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243
Meteorological data
Mean monthly rain fall, relative humidity and tem-
perature were obtained from the Southwestern branch of
Regional Oce of the Ethiopian Meteorological Agency,
Ethiopia.
Ethical considerations
Permission from the district and respective village
authorities for the study was obtained. Both verbal and
written informed consent was obtained from the head of
the households selected for the study for carrying out the
mosquito collection.
Data analysis
Density data were cleaned then log-transformed in
SPSS and also tested for normality before analysis using
SPSS software version 16 (SPSS Inc, Chicago, IL, USA).
The signicance test was done assuming p < 0.05 for the
analysis. Multiple mean comparisons were done by one-
way ANOVA. For signicant ANOVA, means were sepa-
rated using Tukey’s Post-hoc test. Correlation coecient
was used to determine the monthly mean density of An.
gambiae s.l. along with rain fall, relative humidity and
minimum monthly temperature. Student’s t-tests were
used to analyze dierences in indoor and outdoor biting
activities.
Degree of exophily (DE) was calculated as described
by Ameneshewa and Service38: DE = 1−(1/F:HGG) 100,
where, F is the number of fed mosquitoes and HGG is
the sum of the gravid and half-gravid mosquitoes col-
lected by PSCs to determine rates of exophilic behaviour
of collected mosquito species. Parous rates (PR= P/NE),
daily survival rate and longevity (L= 1/–ln p) of
Anopheles mosquitoes were determined following formu-
las developed by Detinova39; where, P=Number of parous
mosquitoes detected, NE=Total number of mosquitoes
examined; and gc=Estimated gonotrophic cycle. Sporo-
gonic cycle and duration of sporogony were calculated
following method developed by WHO36. The sporozoite
infection rate of An. gambiae s.l. was expressed as the
proportion of mosquitoes containing malaria sporozoite
proteins out of the total samples tested by ELISA.
RESULTS
Species compositions and abundance of anopheline
mosquitoes
In total, 1559 adult female anopheline mosquitoes
representing at least three species were collected during
the study period, from the nine study villages (Table 1).
Of these 1122 anopheline mosquitoes were collected by
LTCs and the rest 437 were collected by PSCs. Anopheles
gambiae s.l. (71.8%) was observed as the most abundant
species, followed by An. coustani s.l. (22%) and An. phar-
oensis (6.2%). Anopheles gambiae s.l. and An. coustani
s.l. were found in all the study villages.
Comparison of mosquito density among villages
showed that there was no signicant dierence in mean
density of An. gambiae s.l. (F(8, 117) = 0.32, p = 0.96) and
An. coustani s.l. (F (8, 117) = 0.46, p = 0.89). In contrast,
mean density of An. pharoensis among study villages was
signicantly dierent (F (8, 117) = 8.10, p = 0.0) in distribu-
tion (Table 2).
There was signicant dierence in mean monthly
density of An. gambiae s.l. (F (6, 119) = 84.27, p = 0.0), An.
pharoensis (F (6, 119) = 6.99, p = 0.0) and An. coustani s.l.
(F(6, 119) = 28.58, p = 0.0). The highest mean density of An.
gambiae s.l. was in August, and least in December 2014.
Mean density for An. coustani s.l. was highest during Sep-
tember and October 2014 (Table 3).
Table 1. Species composition and abundance of anophelines
in Kersa district, Jimma zone, southwestern Ethiopia
(June to December 2014)
Species Collection method Total
LTCs PSCs
An. gambiae s.l. 777 (69.25) 342(78.26) 1119 (71.78)
An. pharoensis 56 (5) 42 (9.6) 98 (6.3)
An. coustani s.l. 289 (25.75) 53 (12.14) 342 (21.92)
Total 1122 437 1559
Figures in parentheses indicate number of percentages.
Table 2. Mean density of An. gambiae s.l., An. pharoensis and An.
coustani s.l. (per trap/night) by village in Kersa district, Jimma
zone, southwestern Ethiopia
Kebeles Village An. gambiae s.l. An. pharoensis An. coustani s.l.
(Mean ± SE) (Mean ± SE) (Mean ± SE)
Ankaso Sardo 0.45±0.06
a
0.16±
0.04
a
0.18±0.05
a
Digo 0.40
±0.06
a
0.15±
0.05
a
0.28±0.06
a
Ankaso 0.51
±0.06
a
0.14±0.05
a
0.29±0.07
a
Buko 0.45
±0.07
a
0.0±0.0
b
0.22±0.05
a
Bulbul Waddeyi 0.49±0.07
a
0.0±0.0
b
0.22±0.05
a
Demitu 0.48
±0.08
a
0.0±0.0
b
0.24±0.05
a
Gelo 0.50
±0.08
a
0.0±0.0
b
0.26±0.05
a
Gelo Warsu 0.52
±0.07
a
0.0±0.0
b
0.24±0.05
a
Sayyo 0.46
±0.06
a
0.0±0.0
b
0.21±0.04
a
Means with superscript ‘a’ in the 1st and 3rd column are not signicantly dif-
ferent from each other at p < 0.05. Means with superscript ‘a’ in II column are
signicantly dierent from the means with superscript ‘b’.
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J Vector Borne Dis 54, September 2017
244
Indoor and outdoor density of anopheline mosquitoes
Table 4 shows indoor and outdoor mean monthly den-
sity of An. gambiae s.l., An. pharoensis and An. coustani
s.l. Overall, the mean monthly indoor density of An. gam-
biae s.l. was signicantly higher (t(1,125) = 2.28, p = 0.023)
than the mean monthly outdoor density. However, mean
outdoor density of An. gambiae s.l. was higher than mean
indoor density during September and October 2014.
Degree of exophily of anopheline mosquito
Out of 342 An. gambiae s.l. mosquitoes collected dur-
ing the study period, 196 were fed specimens while 116
were half-gravid and gravid. Higher number of fed and
half-gravid An. gambiae s.l. specimens were documented
in months between June and August and lower degree
of exophily (23–48%) was recorded in the aforemen-
tioned months. Higher degree of exophily was recorded
in months of September (89%) and October (87%), right
after the spray campaign. However, the trend of exophily
was reversed to lower rate in the following months, i.e.
November (56%) and December (50%). Degree of exoph-
ily for An. pharoensis and An. coustani s.l. however, was
higher throughout the intervention period (Table 5).
Daily survival and life expectancy of anopheline mosqui-
toes
Table 6 shows the monthly parity rates, longevity of
An. gambiae s.l. and probability of sporogonic survival
of P. vivax among study villages. Vector mosquitoes
showed longer life span in months June, July and August.
In contrast, they lived shorter life in months September,
October and November. The highest probability of daily
sporogonic survival of P. vivax was observed in June and
August with 0.42 and 0.32 days, respectively, while the
lowest was September (0.03 days).
Table 4. Mean monthly indoor and outdoor anopheline mosquito
density in Kersa district, Jimma zone, southwestern Ethiopia
(June to December 2014)
Months An. gambiae s.l. An. pharoensis An. coustani s.l.
Indoor Outdoor Indoor Outdoor Indoor Outdoor
June 0.67 0.44 0.02 0.2 0.0 0.0
July 0.93 0.67 0.07 0.2 0.04 0.27
August 1.05 0.81 0.0 0.0 0.08 0.44
September 0.30 0.49 0.06 0.2 0.31 0.65
October 0.25 0.32 0.0 0.0 0.21 0.59
November 0.30 0.20 0.0 0.0 0.09 0.49
December 0.12 0.03 0.0 0.0 0.0 0.08
Mean
(%)
0.52
( 5 4 . 7 3 )
0.42
(45.27)
0.02
(20.86)
0.08
(79.14)
0.12
(24.34)
0.36
(75.66)
Number in parentheses indicate percentages of indoor/outdoor species
occurrence.
Table 5. Monthly anopheline abundance and degree of exophily
(DE) recorded in Sarado, Waddeyi and Warsu villages, Kersa
district, southwestern Ethiopia (June to December 2014)
Months Anopheline
species
Abdominal status DE =
1–(1/F:
HGG)
100
Total Unfed Fed HGG F:HGG
Jun An. gambiae s.l. 49 5 29 15 1.9 48
An. pharoensis 16 4 10 2 5 80
Jul An. gambiae s.l. 77 9 44 24 1.8 45
An. pharoensis 14 2 10 2 5 80
Aug An. gambiae s.l. 164 6 89 69 1.3 23
An. coustani s.l. 17 4 11 2 5.5 82
Sep An. gambiae s.l. 15 5 9 1 9 89
An. coustani s.l. 22 4 16 2 8 87
An. pharoensis 12 4 7 1 7 86
Oct An. gambiae s.l. 19 2 15 2 7.5 87
An. coustani s.l. 8 1 6 1 6 83
Nov An. gambiae s.l. 16 3 9 4 2.3 56
An. coustani s.l. 6 1 4 1 4 75
Dec An. gambiae s.l. 2 0 1 1 1 50
F—Number of fed mosquitoes; HGG—The sum of the gravid and half-gravid
mosquitoes.
Infectivity rate of An. gambiae s.l.
Of 192 An. gambiae s.l. tested for Plasmodium para-
site species CSP by sandwich ELISA, two mosquito spec-
imens were found positive for P. vivax 210 polymorphs
with infectivity rate of 1.04%. One of these two positive
specimens was collected in LTCs in the month of June,
while the other was collected in PSCs in August 2014.
Table 3. Mean monthly density/trap-nights of anopheline
mosquitoes in Kersa district, Jimma zone, southwestern Ethiopia
(June to December 2014)
Months An. gambiae s.l.
(Mean ± SE)
An. pharoensis
(Mean ± SE)
An. coustani s.l.
(Mean± SE )
June 0.56±0.04b0.089±0.03a 0.0±0.0e
July 0.81±0.03a 0.13±0.04a 0.16±0.04c,d
August 0.98±0.03a 0.0±0.0b 0.26±0.04b,c
September 0.40±0.04c 0.13±0.04a 0.52±0.04a
October 0.28±0.04c,d 0.0±0.0b 0.40±0.04a
November 0.25±0.03d 0.0±0.0b 0.29±0.04b,c
December 0.08±0.03e 0.0±0.0b 0.04±0.02d,e
Means with the same letter(s) in the same column are not signicantly dier-
ent from each other at p < 0.05; Superscripts refer to the weight of each mean
from highest to lowest starting from ‘a’ to ‘e’.
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245
DISCUSSION
Kersa district in Ethiopia has been reported to be
highly malarious area18, 40. In the present study An. gam-
biae s.l. was recorded from all the study villages and the
highest density was recorded in July and August 2014
which are the months of long rainy season. Rainfall, rela-
tive humidity and temperature favoured the density of An.
gambiae s.l. in the present study, similar to the phenomena
reported in a study in Kenya, which indicated that the rainy
months presents favourable environmental conditions
that enhance mosquito breeding and survival, through the
proliferation of larval habitats and improved humidity41.
Similarly, other studies from Nigeria encountered higher
numbers of mosquitoes in the rainy months42-43.
Anopheles coustani s.l. (some of its sibling species
like An. tenebrosus44 incriminated as suspected second-
ary vectors) was also abundant next to An. gambiae s.l.
in the study villages. The study villages Sarado, Digo and
Ankaso, where An. pharoensis was exclusively recorded
were characterized by permanent water body (Gilgel-
Gibe River, Awetu Rivers, Boy pond, and irrigated areas).
Interestingly, this species which is considered as a sec-
ondary vector in Ethiopia, has been reported from other
similar environments like near Awero and Baro Rivers in
Gambella, southwestern Ethiopia10, 45. This indicates that
this species prefers to breed in permanent water bodies
with emergent vegetation.
Anopheles gambiae s.l. in the present study showed
endophagic behaviour, similar to that report of an ento-
mological assessment in Gambella region10 and Mwea,
Kenya45. In contrast, to the present nding, Kibret et al46,
Woyessa et al47 and Tirados et al48 documented that this
species fed more predominantly in outdoors than indoors
in Zeway, Central Ethiopia, Akaki and in Konso, south-
western Ethiopia respectively. Oyewole et al43 in a study
carried out in Nigeria have also reported that popula-
tion of An. gambiae s.l. species prefer outdoor feeding.
Ameneshewa and Service38 have reported that indoor and
outdoor biting behaviour of An. arabiensis in Gergedi
Upper Awash, central Ethiopia depends strongly on the
availability of hosts. However, in this study, the outdoor
density of An. gambiae s.l., were signicantly higher than
indoor density in September and October 2014 post-IRS
operation. This could be attributed to behavioural avoid-
ance of an insecticide20, 38, 49-50. This higher outdoor density
of the population of An. gambiae s.l. post-IRS operation
may enhance outdoor malaria transmission by increas-
ing human-vector contact, as the local people spend the
evening outdoor with their cattle/villagers or the families
gather together and discuss while chewing Khat (Catha
edulis).
The other two anopheline species documented in
this study (An. pharoensis and An. coustani s.l.) showed
exophagic feeding behaviour, similar to the ndings of
dierent studies from Ethiopia, East Africa and Camer-
oon, West Africa that demonstrated strong exophagic be-
haviour of An. pharoensis and An. coustani s.l.51-54.
Mosquito species varies in their preference of rest-
ing places. The resting behaviour of anophelines has been
shown to be exible and aected by various factors like
vector management methods applied and climate vari-
abilities38, 49, 55. The present study recorded a reduced en-
dophilic preference of An. gambiae s.l. post-IRS interven-
tion. This could be due to irritant and excito-repellency
eect of the insecticide sprayed. In Tanzania, most of the
An. arabiensis were found to exit-trap from DDT-sprayed
houses just after blood meals, compared to houses that
were sprayed with lambdacyhalothrin from which they
left without taking blood meals50. A study conducted in
the Rift Valley of Ethiopia revealed that 43.6% of blood
meal-fed An. arabiensis exiting the DDT sprayed houses
showed exophilic behaviour38. Resting behaviour of vec-
tor is one of the most important factors that determines the
ecacy of IRS. Unfortunately, An. arabiensis, the prin-
cipal vector of malaria in Ethiopia, is partially exophilic
and, thus, poses a greater challenge to malaria control ef-
forts relying on IRS.
During pre-IRS intervention An. gambiae s.l. popula-
tion was observed to show endophilic behaviour. Charl-
wood et al56 in their study carried out in Tanzania and
Maedot et al57 from Eritrea related the increased endo-
philic behaviour of malaria vectors with increased rain-
falls. Fornadel and Norris55 from Zambia indicated that
increased endophily of An. arabiensis could be attributed
to colder or wetter than normal weather which might force
Table 6. Longevity of An. gambiae s.l. and probability of
sporogonic survival of P. vivax in Kersa district, Jimma zone,
southwestern Ethiopia (June to November 2014)
Months Parity P LE (Days) TN (Days) S (Days)
June 0.73 0.9 9.09 27.33 8.18 0.42
July 0.59 0.84 5.88 24.1 10.94 0.15
August 0.71 0.89 8.3 24.92 10.08 0.31
September 0.32 0.68 2.56 26 9.13 0.03
October 0.43 0.76 3.7 27.83 7.88 0.12
November 0.36 0.71 2.94 27.95 7.81 0.07
Mean 0.52 0.79 5.18 26.19 8.98 0.12
P—Probability of daily survival; LE—Life expectancy; T—Atmospheric
temperature; n—Duration of sporogony; S—Probability of daily sporogonic
survival.
Lelisa et al: Anopheline mosquitoes behaviour in southwestern Ethiopia
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J Vector Borne Dis 54, September 2017
246
indoor fed mosquitoes to rest indoor. Long-term use of
DDT in IRS was observed to enhance the behavioural
resistance of this species. The shortest life span of An.
gambiae s.l. in September, suggests that the IRS opera-
tion might have impact on the longevity of the vector spe-
cies. Another possible reason for the low parity rate in late
dry season of the year would be the low temperature and
relative humidity. In the present study, An. gambiae s.l.
showed higher parous rate and longevity during June, July
and August months. Similarly, another study has reported
higher parous rates for An. arabiensis in rainy months38.
The ndings of this study showed that the infectivity
rate of An. gambiae s.l. was low (only 1.04%). Earlier
studies in Ethiopia also indicate low infectivity rates for
the principal malaria vector, An. arabiensis, e.g. sporozo-
ite rates of 1.18 and 1.1% were reported for An. arabiensis
from Sille and Arbaminch city by Habtewold et al58 and
Taye et al52, respectively. Similarly, Oyewole et al43 re-
ported 2.5% sporozoite rate for An. arabiensis in tropical
rain forest of Nigeria. Shililu et al59 also reported 6.3%
sporozoite rates for An. gambiae s.l. in western Kenya.
A CSP negative result in An. gambiae s.l. after IRS in-
tervention might be due to the shorter life expectancy of
the vector species; which implies that, the longer the life
expectancy, the higher the probability of malaria parasite
to reach an infective stage42.
CONCLUSION
In conclusion, the most predominant malaria vector
recorded in the study area is An. gambiae s.l., the principal
malaria vector in Ethiopia. Higher density of An. gambiae
s.l. was recorded outdoor in September month from all
study villages. The fed to gravid ratio of An. gambiae s.l.
was higher after IRS operation; indicating that the spe-
cies tended to avoid the insecticide and rest outdoor due
to induced pressure. Higher density and longevity of An.
gambiae s.l. was observed in the early months of the wet
season (June, July and August) pre-IRS intervention. The
lowest daily survival rates and therefore shorter longevity
of An. gambiae s.l. in September shows the impact of IRS
operation. Thus, the results of the study could contribute
to the understanding of anopheline mosquitoes, i.e. their
composition, dynamics, distribution, life expectancy,
behaviour and infectivity rates in the study area which
could be used in evidence based malaria vector control
programmes.
Conict of interest
The authors declare that they have no any conict of
interests.
ACKNOWLEDGEMENTS
The authors acknowledge the Jimma University,
Jimma, Ethiopia for the nancial support and laboratory
space provision.
REFERENCES
1. World Malaria Report 2016. Geneva: World Health Organiza-
tion 2016. Available from: http://who.int/malaria/publications/
world-malaria-report-2016/report/en. (Accessed on July 9,
2017).
2. World Malaria Report 2015. Geneva, Switzerland: World
Health Organization 2015. Available from: http://who.int/ma-
laria/publications/world-malaria-report-2015/report/en/ (Ac-
cessed on June 4, 2016).
3. Bloland PB. Drug resistance in malaria: A background docu-
ment for the WHO global strategy for containment of antimi-
crobial resistance. WHO/CDS/CSR/DRS/2001.4; Geneva,
Switzerland: World Health Organization 2001; p. 27.
4. Chibsa S. Malaria vector control eorts and challenges in Ethio-
pia. IV Win Meeting, Basel, Switzerland, Oct 24–26, 2007; p.
1–35. Available from: https://pdfs.semanticscholar.org/6a84/5b
a4089d9fa49e7c89861d1bc1fe2108fc0a.pdf (Accessed on June
4, 2016).
5. Guideline for malaria vector control in Ethiopia. Addis Ababa:
Malaria and other Vector Disease Prevention and Control Team,
Disease Prevention and Control Department, Ministry of Health
2002.
6. Corradetti A. The epidemiology of malaria in the region
ullo–jeggiu (Africa oriental Italian). Rev Malar 1940; 2: 101.
7. Giaquinto-Mira. Note on the geographical distribution and biol-
ogy of “Anophilinae” and “Culicinae” in Ethiopia. Riv Malariol
1950; 29(5): 281–313.
8. Fontaine RE, Najjar AE, Prince JS. The 1958 malaria epidemic
in Ethiopia. Am J Trop Med Hyg 1961; 10: 795–803.
9. Rishikesh N. Observations on anopheline vectors of malaria in
an unsprayed upland valley in Ethiopia. World Health Organ
Rep 1966; p. 1–28. Available from: http://apps.who.int/iris/
bitstream/10665/65327/1/WHO_Mal_66.554.pdf (Accessed on
May 3, 2015).
10. Krafsur E. The bionomics and relative prevalence of Anopheles
species with respect to the transmission of Plasmodium to man
in western Ethiopia. J Med Entomol 1977; 14(2): 180–94.
11. Hailemariam LR. Improving eciency, access to and quality of
the rural health extension programme in Tigray, Ethiopia: The
case of malaria diagnosis and treatment. Umeå, Sweden: Print
and Media, Umeå University 2012; p. 48–55.
12. Deressa W, Olana D, Chibsa S. Community participation in
malaria epidemic control in highland areas of southern Oromia,
Ethiopia. Ethiop J Health Develop 2005; 19(1): 3–10.
13. Negash K, Jima D, Nafo-Traore F, Mukelabai K, Banda J,
Medhin A, et al. Ethiopia roll back malaria consultative mis-
sion: Essential actions to support the attainment of the Abuja
targets. Addis Ababa, Ethiopia: Ethiopia RBM Country Consul-
tative Mission Final Report 2004; p. 39.
14. Health sector development programme IV (2010/11-2014/15).
Addis Ababa: Ethiopian Federal Ministry of Health 2010. Avail-
able from: https://www.google.com/url?sa=t&rct=j&q=&esrc=
s&source=web&cd=1&cad=rja&uact=8&ved=0ahUKEwjkm
[Downloaded free from http://www.jvbd.org on Saturday, November 11, 2017, IP: 213.55.106.248]

247
O3zuJjWAhVFNiYKHcQrDCoQFggnMAA&url=http%3A%2F
%2Fwww.nationalplanningcycles.org%2Fsites%2Fdefault%2
Fles%2Fcountry_docs%2FEthiopia%2Fethiopia_hsdp_iv_-
nal_draft_2010_-2015.pdf&usg=AFQjCNFYw6-eT6F6pGGx-
VxXP4M4KbQKSfA; p. 1–114 (Accessed on July 9, 2017).
15. President’s Malaria Initiative. Ethiopia: Malaria Opera-
tional Plan (MOP) FY 2008. Available from: https://www.
google.com/url?sa=t&rct=j&q=&esrc=s&source=web&c
d=1&cad=rja&uact=8&ved=0ahUKEwjIv62DvpjWAhUJ
ORoKHSXrAZcQFggnMAA&url=https%3A%2F%2Fwww.
pmi.gov%2Fdocs%2Fdefault-source%2Fdefault-document-
library%2Fmalaria-operational-plans%2Ffy08%2Fethiopia_
mop-fy08.pdf%3Fsfvrsn%3D6&usg=AFQjCNF-TKIml3uWGt-
8414n5f3Ebftwukg; p. 1–62 (Accessed on July 9, 2017).
16. President’s Malaria Initiative. Ethiopia: Malaria Operational
Plan (MOP) FY 2014. Available from: https://www.usaid.gov/
sites/default/files/documents/1864/Ethiopia%20Malaria%20
Operational%20Plan%20FY%202014.pdf; p. 1–74 (Accessed
on July 9, 2017).
17. Abose T, Yeebiyo Y, Olana D, Alamirew D, Beyene YA, Re-
gassa L, et al. Re-orientation and denition of the role of ma-
laria vector control in Ethiopia: The epidemiology and control
of malaria with special emphasis on the distribution, behaviour
and susceptibility of insecticides of anopheline vectors and
chloroquine resistance in Zwai, central Ethiopia and other areas.
Geneva: World Health Organization 1998. WHO/Mal/1085.
18. Ketema T, Bacha K, Birhanu T, Petros B. Chloroquine-resis-
tant Plasmodium vivax malaria in Serbo Town, Jimma zone,
southwest Ethiopia. Malar J 2009; 8:177.
19. Bloland PB. Drug resistance in malaria: A background
document for the WHO global strategy for containment of
antimicrobial resistance. WHO/CDS/CSR/DRS/2001.4. Geneva,
Switzerland: World Health Organization 2001; p. 27.
20. Yewhalaw D, Wassie F, Steurbaut W, Spanoghe P, Van Bortel W,
Leen Denis, et al. Multiple insecticide resistance: An impedi-
ment to insecticide-based malaria vector control program. PLoS
One 2011; 6(1): e16066. doi:10.1371/journal.pone.0016066.
21. Yewhalaw D, Bortel VW, Denis L, Coosemans M, Duchateau
L, Speybroeck N. First evidence of high knockdown resistance
frequency in Anopheles arabiensis (Diptera: Culicidae) from
Ethiopia. Am J Trop Med Hyg 2010; 3: 122–5.
22. Balkew M, Ibrahim M, Koekemoer LL, Brooke BD, Engers H,
Asea A, et al. Insecticide resistance in Anopheles arabien-
sis (Diptera: Culicidae) from villages in central, northern and
southwest Ethiopia and detection of kdr mutation. Parasit Vec-
tors 2010; 3(40): 3–6.
23. Massebo F, Balkew M, Gebre-Michael T, Lindtjørn B. Blood
meal origins and insecticide susceptibility of Anopheles ara-
biensis from Chano in southwest Ethiopia. Parasit Vectors
2013; 6: 44.
24. Abate A, Hadis M. Susceptibility of Anopheles gambiae s.l. to
DDT, malathion, permethrin and deltamethrin in Ethiopia. Trop
Med Int Health 2011; 16(4): 486–91.
25. Balkew M, Getachew A, Chibsa S, Olana D, Reithinger R,
Brogdon W. Insecticide resistance: A challenge to malaria vec-
tor control in Ethiopia. Malar J 2012; 11(1): 139.
26. Fettene M, Olana D, Christian RN, Koekemoer LL, Coetzee M.
Insecticide resistance in Anopheles arabiensis from Ethiopia.
Afr Entomol 2013; 21(1): 89–94.
27. Yewhalaw D, Asale A, Tushune K, Getachew Y, Duchateau L,
Speybroeck N. Bio-ecacy of selected long-lasting insecticidal
nets against pyrethroid resistant Anopheles arabiensis from
southwestern Ethiopia. Parasit Vectors 2012; 5: 159.
28. Kitau J, Oxborough RM, Tungu PK, Matowo J, Malima RC,
Bruce SJ, et al. Species shifts in the Anopheles gambiae com-
plex: Do LLINs successfully control Anopheles arabiensis?
PLoS One 2012; 7(3): e31481.
29. Gatton ML, Chitnis N, Churcher T, Donnelly MJ, Ghani AC,
Godfray CJ, et al. The importance of mosquito behavioral ad-
aptations to malaria control in Africa. Evolution 2013; 67(4):
1218–30.
30. Moiroux N, Gomez MB, Pennetier C, Elanga E, Djènontin A,
Chandre F, et al. Changes in Anopheles funestus biting behavior
following universal coverage of long-lasting insecticidal nets in
Benin. J Infect Dis 2012; 206(10): 1622–9.
31. Reddy MR, Overgaard HJ, Abaga S, Reddy VP, Caccone A,
Kiszewski AE, et al. Outdoor host seeking behaviour of Anoph-
eles gambiae mosquitoes following initiation of malaria vector
control on Bioko Island, Equatorial Guinea. Malar J 2011; 10:
184.
32. Durnez L, Coosemans M. Residual transmission of malaria: An
old issue for new approaches. In: Prof. Sylvie Manguin, edi-
tor. Anopheles mosquitoes–New insights into malaria vectors.
Croatia: InTech 2016; p. 671–4. Available from: https://www.
intechopen.com/books/anopheles-mosquitoes-new-insights-
into-malaria-vectors/residual-transmission-of-malaria-an-old-
issue-for-new-approaches. doi: 10.5772/55925.
33. Population and housing census of Ethiopia: Administrative
Report, CSA-2012. Addis Ababa: Central Statistics Agency of
Ethiopia 2007; p. 1–125.
34. Gillies M, De Meillon B. The Anophelinae of Africa south of
the Sahara (Ethiopian Zoogeographical Region). Johannesburg:
South African Institute for Medical Research 1968; p. 54.
35. Verrone G. Out-line for the determination of malaria mosquitoes
in Ethiopia. Pt I: Adult female anopheline. Mosq News 1962;
22: 37–49.
36. Manual on practical entomology Pt II: Method and techniques.
Geneva, Switzerland: World Health Organization 1975; p.
1–186.
37. Wirtz R, Zavala F, Charoenvit Y, Campbell G, Burkot T, Schnei-
der I, et al. Comparative testing of monoclonal antibodies
against Plasmodium falciparum sporozoite for ELISA develop-
ment. Bull World Health Organ 1987; 65: 39–45.
38. Ameneshewa B, Service M. Resting habits of Anopheles ara-
biensis in the Awash River Valley of Ethiopia. Ann Trop Med
Parasitol 1996; 90: 515–21.
39. Detinova T. Age-grouping methods in Diptera of medical im-
portance, with special reference to some vectors of malaria.
Monogr Ser World Health Organ 1962; 47: 13–191.
40. Wondimu T, Asea M, Deboch B, Kassahun W. Community
involvement and perception towards malaria prevention and
control strategies in rural areas of Kersa district in Jimma zone,
southwest Ethiopia. Ethiop J Health Develop 2007; 17(1): 1–10.
41. Minakaw N, Sonye G, Mogi M, Githeko A, Yan G. The eects
of climate factors on the distribution and abundance of malaria
vectors in Kenya. J Med Entomol 2002; 39: 833–41.
42. Olayemi I. Survivorship of Anopheles gambiae in relation to
malaria transmission in Ilorin, Nigeria. J Health Allied Sci
2008; 7(3): 1–5.
43. Oyewole I, Awolola T, Ibidapo C, Oduolad A, Okwac O,
Obansad J. Behaviour and population dynamics of the major
anopheline vectors in a malaria endemic area in southern Nige-
ria. J Vector Borne Dis 2007; 44: 56–64.
44. Adugna N, Petros B, Woldegiorgis M, Tilahun D, Lulu M. A
Lelisa et al: Anopheline mosquitoes behaviour in southwestern Ethiopia
[Downloaded free from http://www.jvbd.org on Saturday, November 11, 2017, IP: 213.55.106.248]

J Vector Borne Dis 54, September 2017
248
study of the status of Anopheles tenebrosus (Donitz, 1902) in
the transmission of malaria in Sille, Southern Ethiopia. Ethiop
Med J 1998; 12: 75–80.
45. Muriu MS, Muturi JE, Shililu IJ, Mbogo MC, Mwangangi MJ,
Jacob GB, et al. Host choice and multiple blood feeding be-
haviour of malaria vectors and other anophelines in Mwea rice
scheme, Kenya. Malar J 2008; 7: 43.
46. Kibret S. Petros B, Boelee E, Tekie H. Entomological studies
on the impact of a small-scale irrigation scheme on malar-
ia transmission around Zeway, Ethiopia. In: Awulachew S,
Loulseged M, Yilma A, editor. Impact of irrigation on poverty
and environment in Ethiopia. Proceedings of the Symposium
and Exhibition held in Addis Ababa, November 27–29, 2007;
p. 418–38.
47. Woyessa A, Gebre-Micheal T, Ali A. An indigenous malaria
transmission in the outskirts of Addis Ababa, Akaki Town and
its environs. Ethiop J Health Develop 2004; 18(1): 2–7.
48. Tirados I, Costantini C, Gibson G, Torr S. Blood-feeding behav-
ior of the malarial mosquito Anopheles arabiensis: Implications
for vector control. Med Vet Entomol 2006; 20: 425–37.
49. Fetene M, Hunt RH, Coetzee M, Tessema F. Behavior of Anoph-
eles arabiensis and An. quadriannulatus sp. B mosquitoes and
malaria transmission in southwestern Ethiopia. J Afr Entomol
2004; 12(1): 83–7.
50. Mnzava A, Rwegoshora R, Tanner M, Msuya F, Curtis C, Irare
S. The eects of house spraying with DDT or lambdacyhalo-
thrin against Anopheles arabiensis on measures of malarial
morbidity in children in Tanzania. Acta Trop 1993; 54: 141–51.
51. Antonio-Nkondjio C, Kerah CH, Simard F, Awono-Ambene
P, Chouaibou M, Tchuinkam T, et al. Complexity of malaria
vectorial system in Cameroon: Contribution of secondary
vectors to malaria transmission. J Med Entomol 2006;
43(6): 1215–21.
52. Taye A, Haddis M, Adugna N, Tilahun D, Wirtz R. Biting be-
havior and Plasmodium infection rates of Anopheles arabiensis
from Sille, Ethiopia. Acta Trop 2006; 97: 50–4.
53. Adugna N, Petros B. Determination of the human blood index
of some anopheline mosquitoes by using ELISA. Ethiop Med J
1996; 34: 1–10.
54. Nigatu W, Petros B, Lulu M, Adugna N, Wirtz R. Species com-
position, feeding and resting behavior of the common anthropo-
philic anopheline mosquitoes in relation to malaria transmission
in Gambella, southwest Ethiopia. Int J Trop Insect Sci 1994;
15(3): 371–7.
55. Fornadel CM, Norris DE. Increased endophily by the malaria
vector Anopheles arabiensis in southern Zambia and identica-
tion of digested blood meals. Am J Trop Med Hyg 2008; 79(6):
876–80.
56. Charlwood J, Kihonda S, Sama PF, Billingsley H, Hadji JP, Ver-
have E, et al. The rise and fall of Anopheles arabiensis (Diptera:
Culicidae) in a Tanzanian village. Bull Entomol Res 1995; 85:
37–44.
57. Maedot W, Richard J, Oluyomi A, Chris C. Transmission of
malaria in the Tesseney area of Eritrea: Parasite prevalence in
children, and vector density, host preferences, and sporozoite
rate. J Vet Ecol 2005; 30(1): 27–32.
58. Habtewold T, Walker A, Curtis C, Osir F, Thapa N. The feeding
behavior and Plasmodium infection of Anopheles mosquitoes in
southern Ethiopia in relation to use of insecticide-treated live-
stock for malaria control. Trans R Soc Trop Med Hyg 2001; 95:
584– 6.
59. Shililu J, Maier W, Seitz H, Orago A. Seasonal density
sporozoite rates and entomological inoculation rates of
Anopheles gambiae and Anopheles funustus in high altitude
sugarcane growing zone in western Kenya. Trop Med Int Health
1998; 3(9): 706–10.
Correspondence to: Dr Abebe Asale, Department of Biology, Jimma University, PO Box–5020, Jimma, Ethiopia.
E-mail: abebea663@gmail.com
Received: 9 May 2016 Accepted in revised form: 20 July 2017
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