Group Title: Malaria Journal 2007, 6:50
Title: Larval habitats of Anopheles gambiae s.s. (Diptera: Culicidae) influences vector competence to Plasmodium falciparum parasites
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Title: Larval habitats of Anopheles gambiae s.s. (Diptera: Culicidae) influences vector competence to Plasmodium falciparum parasites
Series Title: Malaria Journal 2007, 6:50
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Creator: Okech BA
Gouagna LC
Yan G
Githure JI
Beier JC
Publication Date: 39202
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alaria Jo nal
Malaria Journal Bilo..e. Cen


Research


Larval habitats of Anopheles gambiae s.s. (Diptera: Culicidae)
influences vector competence to Plasmodium falciparum parasites
Bernard A Okech*1,2,6, Louis C Gouagna2,3, Guiyun Yan5, John I Githure2
and John C Beier4


Address: 'Centre for Biotechnology, Research and Development (CBRD), Kenya Medical Research Institute, P. O. Box 54840, Nairobi, Kenya,
2Human Health Division, International Centre of Insect Physiology and Ecology (ICIPE), P. O. Box 30772, Nairobi, Kenya, 3Departement Societe
et Sante UR 016, Institut de Recherche Pour le Developpement (IRD), P.O. Box 64501, 34394 Montpellier Cedex 5, France, 4Department of
Epidemiology and Public Health, University of Miami School of Medicine, 12500 SW, 152nd Street, Building B Miami, FL 33177, USA, 5Program
in Public Health, College of Health Sciences, University of California, Irvine, Hewitt Hall, Room 3038, Irvine, CA 92697-4050, USA and 6Whitney
Laboratory for Marine Bioscience, University of Florida, 9505 Ocean Shore Boulevard, St Augustine, 32080-8610, FL, USA
Email: Bernard A Okech* okech@whitney.ufl.edu; Louis C Gouagna Louis-Clement.Gouagna@ird.bf; Guiyun Yan guiyuny@uci.edu;
John I Githure jgithure@icipe.org; John C Beier jbeier@med.miami.edu
* Corresponding author



Published: 30 April 2007 Received: 17 January 2007
Malaria journal 2007, 6:50 doi: 10. 1186/1475-2875-6-50 Accepted: 30 April 2007
This article is available from: http://www.malariajournal.com/content/6/l/50
2007 Okech et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.



Abstract
Background: The origin of highly competent malaria vectors has been linked to productive larval
habitats in the field, but there isn't solid quantitative or qualitative data to support it. To test this,
the effect of larval habitat soil substrates on larval development time, pupation rates and vector
competence of Anopheles gambiae to Plasmodium falciparum were examined.
Methods: Soils were collected from active larval habitats with sandy and clay substrates from field
sites and their total organic matter estimated. An. gambiae larvae were reared on these soil
substrates and the larval development time and pupation rates monitored. The emerging adult
mosquitoes were then artificially fed blood with infectious P. falciparum gametocytes from human
volunteers and their midguts examined for oocyst infection after seven days. The wing sizes of the
mosquitoes were also measured. The effect of autoclaving the soil substrates was also evaluated.
Results: The total organic matter was significantly different between clay and sandy soils after
autoclaving (P = 0.022). A generalized liner model (GLM) analysis identified habitat type (clay soil,
sandy soil, or lake water) and autoclaving (that reduces presence of microbes) as significant factors
affecting larval development time and oocyst infection intensities in adults. Autoclaving the soils
resulted in the production of significantly smaller sized mosquitoes (P = 0.008). Autoclaving clay
soils resulted in a significant reduction in Plasmodium falciparum oocyst intensities (P = 0.041) in clay
soils (unautoclaved clay soils (4.28 0.18 oocysts/midgut; autoclaved clay soils = 1.17 0.55
oocysts/midgut) although no difference (P = 0.480) in infection rates was observed between clay
soils (10.4%), sandy soils (5.3%) or lake water (7.9%).
Conclusion: This study suggests an important nutritional role for organic matter and microbial
fauna on mosquito fitness and vector competence. It shows that the quality of natural aquatic
habitats of mosquito larvae may influence malaria parasite transmission potential by An. gambiae.
This information can be important in targeting larval habitats for malaria control.



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Background
Malarial vectors in the Anopheles gambiae complex are
known to use diverse small water bodies as larval habitats
[1]. These habitats differ in physical as well as biological
characteristics, which directly influence the distribution
and abundance of larval mosquito populations in nature
[2]. While it is known from laboratory studies that larval
mosquito nutrition affects vector competence [3,4], the
factors that determine adult An. gambiae fitness for
malaria parasite transmission in the field are unclear, with
only anecdotal evidence suggesting a role for larval habi-
tat productivity [4].

The presence of An. gambiae larvae in small water bodies
has been associated with biotic characteristics such as
plankton, suggesting a contribution by plankton to the
growth and development of the larvae in the field [5,6]. It
has been shown that nutritional resources in larval habi-
tats determine adult mosquito size [3 ], and that a relation-
ship exists between size and parasite infectivity [4], yet no
studies have been conducted to determine if natural mos-
quito larval habitat substrates have an effect on mosquito
productivity or Plasmodium falciparum parasite infectivity
in the adult mosquitoes. The underlying influences of soil
type and organic matter content on larval development,
adult mosquito productivity and on the corresponding
malaria parasite transmission potential of An. gambiae
have not been given much attention.

To answer these questions the effect of different soil sub-
strates on larval development and adult vector compe-
tence of An. gambiae for P falciparum parasites was
evaluated. Soil substrates were sampled from larvae
inhabited water bodies from two geographically isolated
field sites in western Kenya. By using P. falciparum game-
tocytes from human volunteers and An. gambiae reared in
water with natural larval soil substrates, the natural proc-
ess of P. falciparum development in mosquitoes was mim-
icked and thus more light shed on regulatory mechanisms
that have not been well characterized in nature. This study
will enhance the understanding of the nutritional value of
larval mosquito aquatic habitats and their potential influ-
ence on vector competence. Such information may prove
useful for developing malaria control strategies that target
larval habitats.

Materials and methods
Study area
The study was conducted at the International Centre of
Insect Physiology and Ecology (ICIPE) Thomas Odhia-
mbo Campus located in south-western Kenya on the
shores of Lake Victoria, in the Suba District. This area is
surrounded by hills on the south and southeast with the
rest of the area opening up into the low-lying basin facing
the shores of lake. Within the lake itself are several islands;


the nearest is Rusinga, which is joined to the mainland by
a causeway. Stagnant water bodies that make potential
mosquito breeding sites are found on the shores of the
lake. The mosquito breeding habitats in the study area are
diverse. They include small pools, hoof prints, drains,
ditches, river edges, ponds, marshes, man-made holes,
and peri-domestic cemented water containers [7]. The
mean annual rainfall in this area ranges from 1200-1600
mm per year, but rainfall varies by season and year (ICIPE
Thomas Odhiambo Campus meteorological station
data). The long rains usually run from March to June and
the short rains run from October to December. Malaria
transmission in this area is endemic, with An. gambiae s.s.,
An. arabiensis, and An. funestus contributing and sustain-
ing malaria transmission levels estimated at between 0
and 1.55 infectious bites per person per month [8].

The two geographically distinct sites used in this study
were identified based on abundance of larvae, differences
in soil type, and vegetation cover. The first site (Lat: 0,
28.26' S; Long: 34, 17.311' E) near Lwanda Village is a
peninsula in a marshy area with predominantly black cot-
ton soil (clay soil). The site is grazed by many herbivores
(cows, donkeys, sheep, goats, and hippopotami) and is
therefore littered with animal dung and hoof print ponds.
The second site (Lat: 0 24' S; Long: 34 10' E) near
Wasaria Village on Rusinga Island is a well-drained area
with sandy soils that spread over a wide area and wash
into the lake. This area has scattered patches of grass
where herbivores graze (cows, goats and sheep) and fish-
ermen mend nets and sort their fish. In addition to animal
dung and hoof print ponds, fish debris is abundant in the
mosquito aquatic habitats in this area.

Selection and sampling of habitat soils for experiments
To determine which larval habitats to use, the two sites
described above were surveyed to identify productive hab-
itats. A visual inspection of the water bodies was done and
those observed with anopheline larvae were selected. The
densities of larvae in the water bodies were estimated. In
small habitats like hoof print pools, larval densities were
estimated by evacuating the water using a Pasteur pipette
to recover the larvae after which they were counted. Two
larval habitats were selected in each of the study sites
(Lwanda and Rusinga) based on high larval density. Soils
were collected from these habitats at the end of the each
rainy season by digging out the top 10 cm and transport-
ing the soil to the ICIPE Thomas Odhiambo field campus,
where we set up experimental larval habitats. The experi-
mental larval habitats were set up under semi-field condi-
tions inside modified greenhouses [9] to overcome the
many uncontrollable factors in natural conditions, such
as rainfall, other weather conditions, security of sites, co-
habiting predatory organisms that might affect vector pro-
ductivity, and impacts of animals seeking water. The semi-


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field set up allowed us to test many soil samples and to
reliably produce mosquitoes for fitness measurements
and vector competence tests.

Manipulation of soils collected from field sites
The soils were kept in a screen house at the ICIPE-Thomas
Odhiambo campus for approximately two weeks to allow
them to dry out before being used in experiments. This
ensured that any mosquito eggs collected with the soils
would dry up and be rendered non-viable [10,11]. Flood-
ing and exposure to water to hatch any eggs would have
altered the biotic and physico-chemical composition of
the soils and was avoided. The larger particles were broken
into finer pieces and 1,000 cm3 of the soil measured and
spread evenly on the bottom of rearing troughs to make
substrates.

Over a four month period, the sites were visited and soils
collected. A total of six visits were made and after each
visit, the organic matter content of soils was determined
by weighing 10 grams of each soil type that had been
ground and dried and then burning it over a Bunsen
burner at a temperature of 150 C in an open crucible for
three hours. This process converted organic matter to
water and carbon dioxide. The remaining soil material
was then weighed with a micro scale balance. The differ-
ence in the two weights represented the percent loss due
to burning, which directly reflected the total organic mat-
ter in the soil sample. These soil samples were however
not used for rearing mosquitoes. The effect of soil micro-
biota was assessed by autoclaving soil samples at 120C
for 30 minutes to kill microbes and inhibit their activity.
Another soil sample was simply flooded without steriliza-
tion.

Rearing mosquitoes on soil substrates
Gravid colony-reared An. gambiae s.s. mosquitoes (MBITA
strain) were allowed to lay eggs; the eggs were later dis-
pensed into plastic boats and allowed to hatch. Twenty
four hours after hatching, 100 first instar larvae were dis-
pensed into plastic troughs lined with unautoclaved and
autoclaved clay and sandy soil. In each of these experi-
ments, a control experiment was set up with lake water
only. The soil substrates were autoclaved to reduce micro-
bial activity. The first instar larvae were introduced into
equal sized pans (30 x 30 x 5 cm) and their development
monitored. All the troughs were exposed to ambient cli-
mate conditions in the modified greenhouse. No extra
food was given to the developing larvae in the experi-
ments except in the lake water set up. The time for larvae
to develop into pupae (larval development time) and the
number of pupae forming (pupation rate) was recorded
for each rearing pan. Pupae were collected, counted, and
transferred to small cages (15 x 15 x 15 cm) where the
number of emerging adults (emergence rate) was counted.


The adults were provided access to 10% glucose solution
and kept in cages for 3-5 days before being used in exper-
imental infection studies with P. falciparum parasites.

Recruitment of P. falciparum gametocyte carriers
The procedure for recruiting P. falciparum gametocyte pos-
itive human volunteers for experimental infection studies
has been described in detail elsewhere [12]. Briefly, game-
tocytes were obtained from human volunteers recruited
from among patients attending the outpatient department
of the Mbita Health Centre and from villagers and school-
age children from the community surrounding the ICIPE
Thomas Odhiambo Campus. Only gametocyte carriers
within the age group of 3-30 years were recruited, in
accordance with the guidelines of the ethical review
boards of the Kenya Medical Research Institute (KEMRI)
and the University of Miami, USA.

Thick and thin blood smears were collected on slides, and
standard parasitological techniques were employed to
identify P. falciparum asexual and/or sexual parasites.
Only volunteers with observed gametocyte densities
greater than 16 gametocytes per microliter of blood were
selected and used in the study. The goals of the study were
explained to these individuals in a language they could
understand, and individuals over 18 years of age were
requested to sign an informed consent form; if volunteers
were under 18 years, their assent was requested and their
parents or guardians signed the informed consent form on
their behalf.

Experimental infection and assessment of oocyst stages in
mosquitoes
Three to five days after emerging from the soil substrates,
batches of 50-100 female mosquitoes were put in paper
cups, labeled according to the type of habitat soil sub-
strate, and starved for six hours prior to experimental
blood feeding via Parafilm" membrane. The blood for this
experimental infection was obtained by a clinician from
the Mbita Health Centre, who withdrew 2 ml of venous
blood into heparinized tubes from each recruited gameto-
cyte carrier. This blood sample was immediately offered to
mosquitoes in pre-warmed (37oC) artificial membrane
mini-glass feeders. Mosquitoes were allowed to feed for
15 minutes, after which unfed mosquitoes were removed.
The engorged mosquitoes were held at 27 C and 70% rel-
ative humidity. To detect oocysts in the midgut, mosquito
midguts were dissected on day 7 post infection, then
stained with 2% mercurochrome, and each one examined
under a microscope at a magnification of 10x. The wings
of the dissected mosquitoes were mounted on slides and
measured to determine the size of the mosquito [13].






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Statistical analysis
The independent sample T-test (assuming inequality of
variances) was used to determine differences in the total
organic matter between unautoclaved and autoclaved clay
and sandy soils. A generalized linear model (GLM) multi-
variate analysis was used to determine the effects of habi-
tat type and autoclaved soils on larval development time,
number of pupae formed, and number of adults emerg-
ing. GLM univariate analysis was used to test the effect of
the two factors (soil type, autoclaving) on oocyst intensi-
ties in mosquito midguts. The differences in larval devel-
opment time and oocyst intensities were explored further
by Least Significant Difference (LSD) in GLM multivariate
and univariate analysis, respectively, for the different
experimental groups. Pearson's chi square test was applied
to examine the relationships among pupation rates, emer-
gence rates, and soil types. The differences in the mean
oocyst intensities between groups were analysed using
ANOVA and the Tukey test.

Results
The organic content in unautoclaved and autoclaved soils
A total of 6 samples of the soils collected were tested for
organic matter content. There were no significant differ-
ences in the total organic matter between unautoclaved
clay and unautoclaved sandy soils (independent sample
T-test: P = 0.853). In autoclaved soils, a significant differ-
ence was seen in total organic matter content between clay
and sandy soils (T-test: P = 0.022), with more organic
matter observed in clay soils (2.12% 0.09) than in sandy
soils (1.07% 0.39). The process of autoclaving removes
microbes that degrade organic matter, hence a higher
organic matter content was observed after autoclaving.

Effect of larval habitat soils on larval development
The larval development time to pupae in clay soils (5.0
days 0.57) and sandy soils (5.7 days 0.88) were shorter


days 0.33). This trend was similar in autoclaved soils
(Table 1), suggesting that autoclaving (that reduced soil
microbial activity) did not affect larval development time.
However, the larval development time were not signifi-
canty different before (P = 0.606) or after autoclaving (P
= 0.661) although a generalized linear model (GLM) mul-
tivariate analysis identified habitat type as a significant
factor affecting larval development into pupae, with the
replicate as a significant covariant (Table 2). The interac-
tion between habitat type and replicate suggests that tem-
poral changes in the habitat soil composition might affect
larval development parameters. The numbers of pupae
forming and emerging adults were not significantly
affected by habitat type or soil autoclaving (Table 2).
Between un-autoclaved soil types, mosquitoes sizes (clay
soils = 3.134 0.26; and sandy soils = 3.131 0.26) were
not significantly different (ANOVA: df= 1, F =0.010, P =
0.920). However, when the soils were autoclaved, the
sizes of mosquitoes were significantly different (ANOVA:
df= 1, F = 7.161, P = 0.008) between clay (3.012 0.205)
and sandy soils (2.946 0.28). On the other hand, within
soil types, autoclaving did not significantly affect the size
of mosquitoes produced in clay soils (ANOVA: df = 2, F =
0.233, P = 0.796) or in sandy soils (ANOVA: df = 2, F =
0.120, P 0.888).

Effect of larval habitat soils on oocyst infections in
mosquitoes
The oocyst infection rates in mosquitoes reared in clay
soils was (10.4%) slightly higher than in mosquitoes
reared in sandy soils (5.3%) or in control (7.9%) (Table
3). No significant differences in oocyst infection rates
between An. gambiae mosquitoes reared in un-autoclaved
soils were observed (Chi square: X2 = 27.71, df = 28, P =
0.480). A GLM univariate analysis showed that habitat
type and autoclaving soils significantly affected oocyst
intensities in mosquito midguts (Table 4). As expected,


Table I: The influence of autoclaving larval habitat soils on fitness parameters of An. gambiae mosquitoes.

Rearing substrates for larvae


Mosquito parameter measured.

Un-autoclaved soils
Larval development time (d) SD
Pupation rates
Wing sizes SE
Autoclaved soils
Larval development time (d) SD
Pupation rates
Wing size SE


Insectary


6.33 0.33
94.1%
3.12 0.22

6.3 0.33
82.7%
2.85 0.12


Clay soils


5.0 0.57
43.3%
3.08 0.10

5.7 0.88
55.3%
2.94 0.1 I


Sandy soils


5.7 0.88
55.3%
3.16 0.07

5.3 0.88
65.7%
2.86 0.13


Prob.


0.606
0.260
0.523

0.661
0.451
0.641


#Values are means SD of readings from 12 experiments for all the 3 habitat types. In the control group, mosquitoes were reared in lake water
using standard protocols.

compared to mosquito larvae reared in lake water (6.33 the gametocyte density and mosquito size were significant



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Table 2: A GLM multivariate analysis of variance test on the effect of habitat type and autoclaving on An. gambiae mosquito larval
development.


Source of variation


DF Days to pupation


No. of pupae forming6


No of emerging adultsC


F Prob.


Intercept
Site
Treatment
Replicate
Site x Replicate
Site x Treatment x Replicate
Error


* P < 0.05.
Tthe time for larvae to develop to pupae.
Sthe number of pupae forming from a given number of larvae.
the number of adults emerging from a given number of pupae.

factors affecting oocyst intensities (Table 4). The reduc-
tion in oocyst infection intensities (Figure 1) was signifi-
cant in autoclaved clay soils (Tukey Test: P = 0.041) but
not on autoclaved sandy soils (Tukey Test: P = 0.295).

Discussion
This study has demonstrated that productive mosquito
aquatic habitats as detected by soil organic matter content
and microbial activity, contributes to fitness and vector
competence of An. gambiae, which is the major P. falci-
parum malaria transmitter in sub-Saharan Africa. These
two soil parameters may directly contribute to the varia-
tions in malaria parasite transmission seen with this vec-
tor mosquito species in western Kenya [8]. The results of
this study have demonstrated that higher organic matter
and more microbial activity increases mosquito size and
parasite infectivity. The depletion of microbial activity by
autoclaving the soils resulted in reduced food resources
and reduced fitness in the adult mosquitoes. Furthermore,
the reduced oocyst intensities in the midgut of An. gan-


biae mosquitoes reared in autoclaved soils (devoid of
microbes) suggest the influence of soil microbes on the
vector competence of the malaria vector An. gambiae.
Although these results give us an indication about the
influence of habitat soil substrates, several experimental
factors call for caution when interpreting these results.
These factors are the exclusion of some infection experi-
ments due to low mosquito numbers used, zero infected
mosquitoes (Table 3), or the variable number of mosqui-
toes used in experiments (10-30 mosquitoes per group).
However, the difference observed from the effect of soils
was independent of the number of mosquitoes included
in the analysis.

Smaller sized mosquitoes emerging from autoclaved soils
suggests a clear role for soil microbes on larval mosquito
nutrition and development; this is consistent with previ-
ous reports [14,15]. This study suggests that clay soils are
more biologically active, having more nutritive value in
the form of organic matter for the growth and develop-


Table 3: The infectivity of An. gambiae s.l. mosquitoes reared on unautoclaved clay and sandy soil substrates obtained from mosquito
larval habitats in western Kenya and then co-infected with different gametocyte carriers (mean gametocyte density = 173.8 121.4).


Oocyst infection rates


Clay

21.6 (8/37)
9.5 (8/84)
27.3 (12/44)
8.7 (2/23)
0 (0/20)
0 (0/17)
0 (0/40)
0 (0/23)


Oocyst intensity


Sandy

0 (0/56)
0 (0/38)
0(0/51)
0 (0/34)
13.9 (16/1 15)
2.8 (3/108)
16.1 (5/3 1)
0 (0/24)


7.9% (I5/190) 10.4% (30/288) 5.3% (24/457)


Sandy

0
0
0
0
2.0
1.7
2.8
0.0


1.60 0.29 2.06 0.18 1.62 0.59


Only 8 out of 22 experiments successfully yielded infected mosquitoes. The analysis of mosquito infection was done individually to account for
differences in infectiousness among gametocyte carriers and ONLY successful infection experiments have been used in this table.


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I 186.78
2 1 1.65
I 0.000
I 83.51
2 5.559
3 0.000


>0.001
0.001*
0.998
>0.001*
0.017*
0.999


F

34.78
1.67
0.000
1.032
0.647
0.000


Prob.

>0.00 I
0.224
0.998
0.327
0.539
0.999


F.

32.99
0.98
0.000
1.716
0.428
0.000


Prob.

>0.00
0.400
0.998
0.211
0.660
0.999


Gametocyte


Carrier #

I
2
3
4
5
6
7
8


Density

48
16
32
32
128
16
16
16


Lab

15.4 (2/13)
2.2 (1/45)
9.4 (5/53)
ND
40 (2/5)
9.7 (3/31)
6.9 (2/29)
0 (0/14)


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Table 4: A GLM univariate test on the effect of habitat type and treatment of habitat soils by autoclaving on midgut oocyst intensities
in An. gambiae mosquitoes.


Source of variation

Intercept
tHabitat type
STreatment
Habitat type x Treatment
Total organic matter
Gametocyte density
Mosquito size
Error


tHabitat refers to clay or sandy soils mixed with water. STreatment refers to autoclaving or not autoclaving the soils; *Probability, P < 0.05.


ment of mosquitoes. Soil microbiota is responsible for
decomposing organic matter, and their removal through
autoclaving means that the available organic matter in the
soils is not degraded. The products of the organic matter
breakdown increase the amount of organic molecules that
contributes to larval nourishment for growth and survival
[16]. Although soil quality and the corresponding nutri-
tive value may vary between natural breeding sites of An.
gambiae, the lack of major differences in the larval devel-
opment time, number of pupae forming, or number of
emerging adults between unautoclaved and autoclaved
soils suggest that the larvae of An. gambiae can develop
well on non-microbial components of soils. Furthermore,
it shows that larvae of An. gambiae are adaptable to condi-
tions of reduced nutritional quality in their natural habi-
tats. Considering the diversity of water bodies being
utilized by Anopheles larvae in nature [1], it is possible that


Clay soils Sandy soils Control


Figure I
The effect of autoclaving soils from clay and sandy larval hab-
itats on the oocyst infection intensities in the midguts of An.
gambiae mosquitoes.


larvae may survive on dissolved organic and inorganic
molecules [6,16]. The results herein provide important
clues of other potential food resources available for
Anopheles mosquito larvae in natural water bodies.

The response of An. gambiae mosquitoes to malaria para-
site infection varies as a function of habitat type, biologi-
cal activity of the habitat, size of the mosquito, and
gametocyte density. This study determined that soil
organic matter content was different between habitat
types and may have directly influenced the sizes of the
mosquitoes produced. Although other factors, such as
temperature [17] or larval densities of larvae in a habitat
[17], may also affect the size of mosquitoes produced,
these were controlled for in the experimental setup and
therefore did not contribute to the differences observed.
The size of the mosquito may influence oocyst intensities
in the midgut of naturally infected mosquitoes [4]. The
observed high organic matter content in clay soils, may
have led to the production of larger mosquitoes. This in
turn led to higher oocyst intensities in mosquitoes reared
in predominantly clay habitats. Water bodies on clay soils
appear turbid because of the presence of particulate mat-
ter in colloidal suspension. To quantify the amount or
identity of the particulate matter in clay habitats was not
logistically possible in this study. However, Anopheles
mosquito larvae may feed through their suspension feed-
ing mechanism on such particles to derive extra nutriment
[16]. This extra nutriment could have led to the higher fit-
ness observed in emerging adult mosquitoes on clay soils.

The influence of natural larval habitats on An. gambiae fit-
ness and vector competence should be carefully consid-
ered to understand the ecology of this important malaria
vector. Furthermore the effect of the factors identified in
this study, as well as other unknown or uncontrolled fac-
tors in larval aquatic habitats, may be part of the system
that determines the heterogeneity of larval developmental
stages in water bodies and adult vector mosquito produc-
tion in nature. In urban environments, it is assumed that
environmental pollutants are likely to reduce mosquito


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MS

1.275
0.978
1.214
0.664
0.401
1.580
1.193
0.198


6.423
4.928
6.120
3.347
2.018
7.965
6.01 1


0.022
0.041*
0.025*
0.086
0.175
0.012*
0.026*


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fitness (Paul Miregi, pers. comm.) and vector competence.
As demonstrated in this study, changes in habitat quality
due to microbial transformation or in the organic matter
content may directly govern the distribution of Anopheles
mosquitoes and the risk of malaria transmission in
endemic areas. Inherently, it is of value to identify such
productive larval habitats in nature as their removal will
greatly contribute to malaria control efforts.


Authors' contributions
BAO Designed experiments, collected and analyzed
data, wrote manuscript; LCG Data collection and review
of manuscript; JCB Review of data and manuscript; GY
- Review of manuscript; JIG Administrative support and
review of manuscript. All authors have read and agree
with the contents in the paper.


Acknowledgements
All the ICIPE Thomas Odhiambo Campus staff members, particularly the
vector competence project team members, for their input in this study.
Also appreciation to the local chiefs, clan elders, village communities,
schools, and head teachers for the cooperation received from them in the
recruitment of gametocyte carriers. Many thanks to the personnel of the
Mbita Health Centre for their support. This study was supported by NIH
grants U19A14551 I, D43TWOI 143, D43TW00920 and D43TW01505.
BAO received an African Regional Postgraduate Programme in Insect Sci-
ence (ARPPIS) training fellowship from ICIPE. This paper has been pub-
lished with the permission of the Director, Kenya Medical Research
Institute (KEMRI).

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S, BierJC, Knols BG, GithureJl, Yan G: Infectivity of Plasmodium
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Malaria Joumnal 2007, 6:50







alaria Jo nal
Malaria Journal Bilo..e. Cen


Research


Larval habitats of Anopheles gambiae s.s. (Diptera: Culicidae)
influences vector competence to Plasmodium falciparum parasites
Bernard A Okech*1,2,6, Louis C Gouagna2,3, Guiyun Yan5, John I Githure2
and John C Beier4


Address: 'Centre for Biotechnology, Research and Development (CBRD), Kenya Medical Research Institute, P. O. Box 54840, Nairobi, Kenya,
2Human Health Division, International Centre of Insect Physiology and Ecology (ICIPE), P. O. Box 30772, Nairobi, Kenya, 3Departement Societe
et Sante UR 016, Institut de Recherche Pour le Developpement (IRD), P.O. Box 64501, 34394 Montpellier Cedex 5, France, 4Department of
Epidemiology and Public Health, University of Miami School of Medicine, 12500 SW, 152nd Street, Building B Miami, FL 33177, USA, 5Program
in Public Health, College of Health Sciences, University of California, Irvine, Hewitt Hall, Room 3038, Irvine, CA 92697-4050, USA and 6Whitney
Laboratory for Marine Bioscience, University of Florida, 9505 Ocean Shore Boulevard, St Augustine, 32080-8610, FL, USA
Email: Bernard A Okech* okech@whitney.ufl.edu; Louis C Gouagna Louis-Clement.Gouagna@ird.bf; Guiyun Yan guiyuny@uci.edu;
John I Githure jgithure@icipe.org; John C Beier jbeier@med.miami.edu
* Corresponding author



Published: 30 April 2007 Received: 17 January 2007
Malaria journal 2007, 6:50 doi: 10. 1186/1475-2875-6-50 Accepted: 30 April 2007
This article is available from: http://www.malariajournal.com/content/6/l/50
2007 Okech et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.



Abstract
Background: The origin of highly competent malaria vectors has been linked to productive larval
habitats in the field, but there isn't solid quantitative or qualitative data to support it. To test this,
the effect of larval habitat soil substrates on larval development time, pupation rates and vector
competence of Anopheles gambiae to Plasmodium falciparum were examined.
Methods: Soils were collected from active larval habitats with sandy and clay substrates from field
sites and their total organic matter estimated. An. gambiae larvae were reared on these soil
substrates and the larval development time and pupation rates monitored. The emerging adult
mosquitoes were then artificially fed blood with infectious P. falciparum gametocytes from human
volunteers and their midguts examined for oocyst infection after seven days. The wing sizes of the
mosquitoes were also measured. The effect of autoclaving the soil substrates was also evaluated.
Results: The total organic matter was significantly different between clay and sandy soils after
autoclaving (P = 0.022). A generalized liner model (GLM) analysis identified habitat type (clay soil,
sandy soil, or lake water) and autoclaving (that reduces presence of microbes) as significant factors
affecting larval development time and oocyst infection intensities in adults. Autoclaving the soils
resulted in the production of significantly smaller sized mosquitoes (P = 0.008). Autoclaving clay
soils resulted in a significant reduction in Plasmodium falciparum oocyst intensities (P = 0.041) in clay
soils (unautoclaved clay soils (4.28 0.18 oocysts/midgut; autoclaved clay soils = 1.17 0.55
oocysts/midgut) although no difference (P = 0.480) in infection rates was observed between clay
soils (10.4%), sandy soils (5.3%) or lake water (7.9%).
Conclusion: This study suggests an important nutritional role for organic matter and microbial
fauna on mosquito fitness and vector competence. It shows that the quality of natural aquatic
habitats of mosquito larvae may influence malaria parasite transmission potential by An. gambiae.
This information can be important in targeting larval habitats for malaria control.



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Background
Malarial vectors in the Anopheles gambiae complex are
known to use diverse small water bodies as larval habitats
[1]. These habitats differ in physical as well as biological
characteristics, which directly influence the distribution
and abundance of larval mosquito populations in nature
[2]. While it is known from laboratory studies that larval
mosquito nutrition affects vector competence [3,4], the
factors that determine adult An. gambiae fitness for
malaria parasite transmission in the field are unclear, with
only anecdotal evidence suggesting a role for larval habi-
tat productivity [4].

The presence of An. gambiae larvae in small water bodies
has been associated with biotic characteristics such as
plankton, suggesting a contribution by plankton to the
growth and development of the larvae in the field [5,6]. It
has been shown that nutritional resources in larval habi-
tats determine adult mosquito size [3 ], and that a relation-
ship exists between size and parasite infectivity [4], yet no
studies have been conducted to determine if natural mos-
quito larval habitat substrates have an effect on mosquito
productivity or Plasmodium falciparum parasite infectivity
in the adult mosquitoes. The underlying influences of soil
type and organic matter content on larval development,
adult mosquito productivity and on the corresponding
malaria parasite transmission potential of An. gambiae
have not been given much attention.

To answer these questions the effect of different soil sub-
strates on larval development and adult vector compe-
tence of An. gambiae for P falciparum parasites was
evaluated. Soil substrates were sampled from larvae
inhabited water bodies from two geographically isolated
field sites in western Kenya. By using P. falciparum game-
tocytes from human volunteers and An. gambiae reared in
water with natural larval soil substrates, the natural proc-
ess of P. falciparum development in mosquitoes was mim-
icked and thus more light shed on regulatory mechanisms
that have not been well characterized in nature. This study
will enhance the understanding of the nutritional value of
larval mosquito aquatic habitats and their potential influ-
ence on vector competence. Such information may prove
useful for developing malaria control strategies that target
larval habitats.

Materials and methods
Study area
The study was conducted at the International Centre of
Insect Physiology and Ecology (ICIPE) Thomas Odhia-
mbo Campus located in south-western Kenya on the
shores of Lake Victoria, in the Suba District. This area is
surrounded by hills on the south and southeast with the
rest of the area opening up into the low-lying basin facing
the shores of lake. Within the lake itself are several islands;


the nearest is Rusinga, which is joined to the mainland by
a causeway. Stagnant water bodies that make potential
mosquito breeding sites are found on the shores of the
lake. The mosquito breeding habitats in the study area are
diverse. They include small pools, hoof prints, drains,
ditches, river edges, ponds, marshes, man-made holes,
and peri-domestic cemented water containers [7]. The
mean annual rainfall in this area ranges from 1200-1600
mm per year, but rainfall varies by season and year (ICIPE
Thomas Odhiambo Campus meteorological station
data). The long rains usually run from March to June and
the short rains run from October to December. Malaria
transmission in this area is endemic, with An. gambiae s.s.,
An. arabiensis, and An. funestus contributing and sustain-
ing malaria transmission levels estimated at between 0
and 1.55 infectious bites per person per month [8].

The two geographically distinct sites used in this study
were identified based on abundance of larvae, differences
in soil type, and vegetation cover. The first site (Lat: 0,
28.26' S; Long: 34, 17.311' E) near Lwanda Village is a
peninsula in a marshy area with predominantly black cot-
ton soil (clay soil). The site is grazed by many herbivores
(cows, donkeys, sheep, goats, and hippopotami) and is
therefore littered with animal dung and hoof print ponds.
The second site (Lat: 0 24' S; Long: 34 10' E) near
Wasaria Village on Rusinga Island is a well-drained area
with sandy soils that spread over a wide area and wash
into the lake. This area has scattered patches of grass
where herbivores graze (cows, goats and sheep) and fish-
ermen mend nets and sort their fish. In addition to animal
dung and hoof print ponds, fish debris is abundant in the
mosquito aquatic habitats in this area.

Selection and sampling of habitat soils for experiments
To determine which larval habitats to use, the two sites
described above were surveyed to identify productive hab-
itats. A visual inspection of the water bodies was done and
those observed with anopheline larvae were selected. The
densities of larvae in the water bodies were estimated. In
small habitats like hoof print pools, larval densities were
estimated by evacuating the water using a Pasteur pipette
to recover the larvae after which they were counted. Two
larval habitats were selected in each of the study sites
(Lwanda and Rusinga) based on high larval density. Soils
were collected from these habitats at the end of the each
rainy season by digging out the top 10 cm and transport-
ing the soil to the ICIPE Thomas Odhiambo field campus,
where we set up experimental larval habitats. The experi-
mental larval habitats were set up under semi-field condi-
tions inside modified greenhouses [9] to overcome the
many uncontrollable factors in natural conditions, such
as rainfall, other weather conditions, security of sites, co-
habiting predatory organisms that might affect vector pro-
ductivity, and impacts of animals seeking water. The semi-


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field set up allowed us to test many soil samples and to
reliably produce mosquitoes for fitness measurements
and vector competence tests.

Manipulation of soils collected from field sites
The soils were kept in a screen house at the ICIPE-Thomas
Odhiambo campus for approximately two weeks to allow
them to dry out before being used in experiments. This
ensured that any mosquito eggs collected with the soils
would dry up and be rendered non-viable [10,11]. Flood-
ing and exposure to water to hatch any eggs would have
altered the biotic and physico-chemical composition of
the soils and was avoided. The larger particles were broken
into finer pieces and 1,000 cm3 of the soil measured and
spread evenly on the bottom of rearing troughs to make
substrates.

Over a four month period, the sites were visited and soils
collected. A total of six visits were made and after each
visit, the organic matter content of soils was determined
by weighing 10 grams of each soil type that had been
ground and dried and then burning it over a Bunsen
burner at a temperature of 150 C in an open crucible for
three hours. This process converted organic matter to
water and carbon dioxide. The remaining soil material
was then weighed with a micro scale balance. The differ-
ence in the two weights represented the percent loss due
to burning, which directly reflected the total organic mat-
ter in the soil sample. These soil samples were however
not used for rearing mosquitoes. The effect of soil micro-
biota was assessed by autoclaving soil samples at 120C
for 30 minutes to kill microbes and inhibit their activity.
Another soil sample was simply flooded without steriliza-
tion.

Rearing mosquitoes on soil substrates
Gravid colony-reared An. gambiae s.s. mosquitoes (MBITA
strain) were allowed to lay eggs; the eggs were later dis-
pensed into plastic boats and allowed to hatch. Twenty
four hours after hatching, 100 first instar larvae were dis-
pensed into plastic troughs lined with unautoclaved and
autoclaved clay and sandy soil. In each of these experi-
ments, a control experiment was set up with lake water
only. The soil substrates were autoclaved to reduce micro-
bial activity. The first instar larvae were introduced into
equal sized pans (30 x 30 x 5 cm) and their development
monitored. All the troughs were exposed to ambient cli-
mate conditions in the modified greenhouse. No extra
food was given to the developing larvae in the experi-
ments except in the lake water set up. The time for larvae
to develop into pupae (larval development time) and the
number of pupae forming (pupation rate) was recorded
for each rearing pan. Pupae were collected, counted, and
transferred to small cages (15 x 15 x 15 cm) where the
number of emerging adults (emergence rate) was counted.


The adults were provided access to 10% glucose solution
and kept in cages for 3-5 days before being used in exper-
imental infection studies with P. falciparum parasites.

Recruitment of P. falciparum gametocyte carriers
The procedure for recruiting P. falciparum gametocyte pos-
itive human volunteers for experimental infection studies
has been described in detail elsewhere [12]. Briefly, game-
tocytes were obtained from human volunteers recruited
from among patients attending the outpatient department
of the Mbita Health Centre and from villagers and school-
age children from the community surrounding the ICIPE
Thomas Odhiambo Campus. Only gametocyte carriers
within the age group of 3-30 years were recruited, in
accordance with the guidelines of the ethical review
boards of the Kenya Medical Research Institute (KEMRI)
and the University of Miami, USA.

Thick and thin blood smears were collected on slides, and
standard parasitological techniques were employed to
identify P. falciparum asexual and/or sexual parasites.
Only volunteers with observed gametocyte densities
greater than 16 gametocytes per microliter of blood were
selected and used in the study. The goals of the study were
explained to these individuals in a language they could
understand, and individuals over 18 years of age were
requested to sign an informed consent form; if volunteers
were under 18 years, their assent was requested and their
parents or guardians signed the informed consent form on
their behalf.

Experimental infection and assessment of oocyst stages in
mosquitoes
Three to five days after emerging from the soil substrates,
batches of 50-100 female mosquitoes were put in paper
cups, labeled according to the type of habitat soil sub-
strate, and starved for six hours prior to experimental
blood feeding via Parafilm" membrane. The blood for this
experimental infection was obtained by a clinician from
the Mbita Health Centre, who withdrew 2 ml of venous
blood into heparinized tubes from each recruited gameto-
cyte carrier. This blood sample was immediately offered to
mosquitoes in pre-warmed (37oC) artificial membrane
mini-glass feeders. Mosquitoes were allowed to feed for
15 minutes, after which unfed mosquitoes were removed.
The engorged mosquitoes were held at 27 C and 70% rel-
ative humidity. To detect oocysts in the midgut, mosquito
midguts were dissected on day 7 post infection, then
stained with 2% mercurochrome, and each one examined
under a microscope at a magnification of 10x. The wings
of the dissected mosquitoes were mounted on slides and
measured to determine the size of the mosquito [13].






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Statistical analysis
The independent sample T-test (assuming inequality of
variances) was used to determine differences in the total
organic matter between unautoclaved and autoclaved clay
and sandy soils. A generalized linear model (GLM) multi-
variate analysis was used to determine the effects of habi-
tat type and autoclaved soils on larval development time,
number of pupae formed, and number of adults emerg-
ing. GLM univariate analysis was used to test the effect of
the two factors (soil type, autoclaving) on oocyst intensi-
ties in mosquito midguts. The differences in larval devel-
opment time and oocyst intensities were explored further
by Least Significant Difference (LSD) in GLM multivariate
and univariate analysis, respectively, for the different
experimental groups. Pearson's chi square test was applied
to examine the relationships among pupation rates, emer-
gence rates, and soil types. The differences in the mean
oocyst intensities between groups were analysed using
ANOVA and the Tukey test.

Results
The organic content in unautoclaved and autoclaved soils
A total of 6 samples of the soils collected were tested for
organic matter content. There were no significant differ-
ences in the total organic matter between unautoclaved
clay and unautoclaved sandy soils (independent sample
T-test: P = 0.853). In autoclaved soils, a significant differ-
ence was seen in total organic matter content between clay
and sandy soils (T-test: P = 0.022), with more organic
matter observed in clay soils (2.12% 0.09) than in sandy
soils (1.07% 0.39). The process of autoclaving removes
microbes that degrade organic matter, hence a higher
organic matter content was observed after autoclaving.

Effect of larval habitat soils on larval development
The larval development time to pupae in clay soils (5.0
days 0.57) and sandy soils (5.7 days 0.88) were shorter


days 0.33). This trend was similar in autoclaved soils
(Table 1), suggesting that autoclaving (that reduced soil
microbial activity) did not affect larval development time.
However, the larval development time were not signifi-
canty different before (P = 0.606) or after autoclaving (P
= 0.661) although a generalized linear model (GLM) mul-
tivariate analysis identified habitat type as a significant
factor affecting larval development into pupae, with the
replicate as a significant covariant (Table 2). The interac-
tion between habitat type and replicate suggests that tem-
poral changes in the habitat soil composition might affect
larval development parameters. The numbers of pupae
forming and emerging adults were not significantly
affected by habitat type or soil autoclaving (Table 2).
Between un-autoclaved soil types, mosquitoes sizes (clay
soils = 3.134 0.26; and sandy soils = 3.131 0.26) were
not significantly different (ANOVA: df= 1, F =0.010, P =
0.920). However, when the soils were autoclaved, the
sizes of mosquitoes were significantly different (ANOVA:
df= 1, F = 7.161, P = 0.008) between clay (3.012 0.205)
and sandy soils (2.946 0.28). On the other hand, within
soil types, autoclaving did not significantly affect the size
of mosquitoes produced in clay soils (ANOVA: df = 2, F =
0.233, P = 0.796) or in sandy soils (ANOVA: df = 2, F =
0.120, P 0.888).

Effect of larval habitat soils on oocyst infections in
mosquitoes
The oocyst infection rates in mosquitoes reared in clay
soils was (10.4%) slightly higher than in mosquitoes
reared in sandy soils (5.3%) or in control (7.9%) (Table
3). No significant differences in oocyst infection rates
between An. gambiae mosquitoes reared in un-autoclaved
soils were observed (Chi square: X2 = 27.71, df = 28, P =
0.480). A GLM univariate analysis showed that habitat
type and autoclaving soils significantly affected oocyst
intensities in mosquito midguts (Table 4). As expected,


Table I: The influence of autoclaving larval habitat soils on fitness parameters of An. gambiae mosquitoes.

Rearing substrates for larvae


Mosquito parameter measured.

Un-autoclaved soils
Larval development time (d) SD
Pupation rates
Wing sizes SE
Autoclaved soils
Larval development time (d) SD
Pupation rates
Wing size SE


Insectary


6.33 0.33
94.1%
3.12 0.22

6.3 0.33
82.7%
2.85 0.12


Clay soils


5.0 0.57
43.3%
3.08 0.10

5.7 0.88
55.3%
2.94 0.1 I


Sandy soils


5.7 0.88
55.3%
3.16 0.07

5.3 0.88
65.7%
2.86 0.13


Prob.


0.606
0.260
0.523

0.661
0.451
0.641


#Values are means SD of readings from 12 experiments for all the 3 habitat types. In the control group, mosquitoes were reared in lake water
using standard protocols.

compared to mosquito larvae reared in lake water (6.33 the gametocyte density and mosquito size were significant



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Table 2: A GLM multivariate analysis of variance test on the effect of habitat type and autoclaving on An. gambiae mosquito larval
development.


Source of variation


DF Days to pupation


No. of pupae forming6


No of emerging adultsC


F Prob.


Intercept
Site
Treatment
Replicate
Site x Replicate
Site x Treatment x Replicate
Error


* P < 0.05.
Tthe time for larvae to develop to pupae.
Sthe number of pupae forming from a given number of larvae.
the number of adults emerging from a given number of pupae.

factors affecting oocyst intensities (Table 4). The reduc-
tion in oocyst infection intensities (Figure 1) was signifi-
cant in autoclaved clay soils (Tukey Test: P = 0.041) but
not on autoclaved sandy soils (Tukey Test: P = 0.295).

Discussion
This study has demonstrated that productive mosquito
aquatic habitats as detected by soil organic matter content
and microbial activity, contributes to fitness and vector
competence of An. gambiae, which is the major P. falci-
parum malaria transmitter in sub-Saharan Africa. These
two soil parameters may directly contribute to the varia-
tions in malaria parasite transmission seen with this vec-
tor mosquito species in western Kenya [8]. The results of
this study have demonstrated that higher organic matter
and more microbial activity increases mosquito size and
parasite infectivity. The depletion of microbial activity by
autoclaving the soils resulted in reduced food resources
and reduced fitness in the adult mosquitoes. Furthermore,
the reduced oocyst intensities in the midgut of An. gan-


biae mosquitoes reared in autoclaved soils (devoid of
microbes) suggest the influence of soil microbes on the
vector competence of the malaria vector An. gambiae.
Although these results give us an indication about the
influence of habitat soil substrates, several experimental
factors call for caution when interpreting these results.
These factors are the exclusion of some infection experi-
ments due to low mosquito numbers used, zero infected
mosquitoes (Table 3), or the variable number of mosqui-
toes used in experiments (10-30 mosquitoes per group).
However, the difference observed from the effect of soils
was independent of the number of mosquitoes included
in the analysis.

Smaller sized mosquitoes emerging from autoclaved soils
suggests a clear role for soil microbes on larval mosquito
nutrition and development; this is consistent with previ-
ous reports [14,15]. This study suggests that clay soils are
more biologically active, having more nutritive value in
the form of organic matter for the growth and develop-


Table 3: The infectivity of An. gambiae s.l. mosquitoes reared on unautoclaved clay and sandy soil substrates obtained from mosquito
larval habitats in western Kenya and then co-infected with different gametocyte carriers (mean gametocyte density = 173.8 121.4).


Oocyst infection rates


Clay

21.6 (8/37)
9.5 (8/84)
27.3 (12/44)
8.7 (2/23)
0 (0/20)
0 (0/17)
0 (0/40)
0 (0/23)


Oocyst intensity


Sandy

0 (0/56)
0 (0/38)
0(0/51)
0 (0/34)
13.9 (16/1 15)
2.8 (3/108)
16.1 (5/3 1)
0 (0/24)


7.9% (I5/190) 10.4% (30/288) 5.3% (24/457)


Sandy

0
0
0
0
2.0
1.7
2.8
0.0


1.60 0.29 2.06 0.18 1.62 0.59


Only 8 out of 22 experiments successfully yielded infected mosquitoes. The analysis of mosquito infection was done individually to account for
differences in infectiousness among gametocyte carriers and ONLY successful infection experiments have been used in this table.


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I 186.78
2 1 1.65
I 0.000
I 83.51
2 5.559
3 0.000


>0.001
0.001*
0.998
>0.001*
0.017*
0.999


F

34.78
1.67
0.000
1.032
0.647
0.000


Prob.

>0.00 I
0.224
0.998
0.327
0.539
0.999


F.

32.99
0.98
0.000
1.716
0.428
0.000


Prob.

>0.00
0.400
0.998
0.211
0.660
0.999


Gametocyte


Carrier #

I
2
3
4
5
6
7
8


Density

48
16
32
32
128
16
16
16


Lab

15.4 (2/13)
2.2 (1/45)
9.4 (5/53)
ND
40 (2/5)
9.7 (3/31)
6.9 (2/29)
0 (0/14)


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Table 4: A GLM univariate test on the effect of habitat type and treatment of habitat soils by autoclaving on midgut oocyst intensities
in An. gambiae mosquitoes.


Source of variation

Intercept
tHabitat type
STreatment
Habitat type x Treatment
Total organic matter
Gametocyte density
Mosquito size
Error


tHabitat refers to clay or sandy soils mixed with water. STreatment refers to autoclaving or not autoclaving the soils; *Probability, P < 0.05.


ment of mosquitoes. Soil microbiota is responsible for
decomposing organic matter, and their removal through
autoclaving means that the available organic matter in the
soils is not degraded. The products of the organic matter
breakdown increase the amount of organic molecules that
contributes to larval nourishment for growth and survival
[16]. Although soil quality and the corresponding nutri-
tive value may vary between natural breeding sites of An.
gambiae, the lack of major differences in the larval devel-
opment time, number of pupae forming, or number of
emerging adults between unautoclaved and autoclaved
soils suggest that the larvae of An. gambiae can develop
well on non-microbial components of soils. Furthermore,
it shows that larvae of An. gambiae are adaptable to condi-
tions of reduced nutritional quality in their natural habi-
tats. Considering the diversity of water bodies being
utilized by Anopheles larvae in nature [1], it is possible that


Clay soils Sandy soils Control


Figure I
The effect of autoclaving soils from clay and sandy larval hab-
itats on the oocyst infection intensities in the midguts of An.
gambiae mosquitoes.


larvae may survive on dissolved organic and inorganic
molecules [6,16]. The results herein provide important
clues of other potential food resources available for
Anopheles mosquito larvae in natural water bodies.

The response of An. gambiae mosquitoes to malaria para-
site infection varies as a function of habitat type, biologi-
cal activity of the habitat, size of the mosquito, and
gametocyte density. This study determined that soil
organic matter content was different between habitat
types and may have directly influenced the sizes of the
mosquitoes produced. Although other factors, such as
temperature [17] or larval densities of larvae in a habitat
[17], may also affect the size of mosquitoes produced,
these were controlled for in the experimental setup and
therefore did not contribute to the differences observed.
The size of the mosquito may influence oocyst intensities
in the midgut of naturally infected mosquitoes [4]. The
observed high organic matter content in clay soils, may
have led to the production of larger mosquitoes. This in
turn led to higher oocyst intensities in mosquitoes reared
in predominantly clay habitats. Water bodies on clay soils
appear turbid because of the presence of particulate mat-
ter in colloidal suspension. To quantify the amount or
identity of the particulate matter in clay habitats was not
logistically possible in this study. However, Anopheles
mosquito larvae may feed through their suspension feed-
ing mechanism on such particles to derive extra nutriment
[16]. This extra nutriment could have led to the higher fit-
ness observed in emerging adult mosquitoes on clay soils.

The influence of natural larval habitats on An. gambiae fit-
ness and vector competence should be carefully consid-
ered to understand the ecology of this important malaria
vector. Furthermore the effect of the factors identified in
this study, as well as other unknown or uncontrolled fac-
tors in larval aquatic habitats, may be part of the system
that determines the heterogeneity of larval developmental
stages in water bodies and adult vector mosquito produc-
tion in nature. In urban environments, it is assumed that
environmental pollutants are likely to reduce mosquito


Page 6 of 7
(page number not for citation purposes)


MS

1.275
0.978
1.214
0.664
0.401
1.580
1.193
0.198


6.423
4.928
6.120
3.347
2.018
7.965
6.01 1


0.022
0.041*
0.025*
0.086
0.175
0.012*
0.026*


Malaria Journal 2007, 6:50








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fitness (Paul Miregi, pers. comm.) and vector competence.
As demonstrated in this study, changes in habitat quality
due to microbial transformation or in the organic matter
content may directly govern the distribution of Anopheles
mosquitoes and the risk of malaria transmission in
endemic areas. Inherently, it is of value to identify such
productive larval habitats in nature as their removal will
greatly contribute to malaria control efforts.


Authors' contributions
BAO Designed experiments, collected and analyzed
data, wrote manuscript; LCG Data collection and review
of manuscript; JCB Review of data and manuscript; GY
- Review of manuscript; JIG Administrative support and
review of manuscript. All authors have read and agree
with the contents in the paper.


Acknowledgements
All the ICIPE Thomas Odhiambo Campus staff members, particularly the
vector competence project team members, for their input in this study.
Also appreciation to the local chiefs, clan elders, village communities,
schools, and head teachers for the cooperation received from them in the
recruitment of gametocyte carriers. Many thanks to the personnel of the
Mbita Health Centre for their support. This study was supported by NIH
grants U19A14551 I, D43TWOI 143, D43TW00920 and D43TW01505.
BAO received an African Regional Postgraduate Programme in Insect Sci-
ence (ARPPIS) training fellowship from ICIPE. This paper has been pub-
lished with the permission of the Director, Kenya Medical Research
Institute (KEMRI).

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