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Land Use Change and the Microbial Ecology of Anopheles gambiae✔
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Thomas M. Gilbreath III, Orange County Vector Control District13001Garden Grove Blvd, Garden Grove, California 92843, [email protected], tel:+1(949)943-9720
Guofa Zhou, Program in Public Health,University of California, Irvine3501 Hewitt Hall, Irvine, California 92697, [email protected], tel:+1(949)824-0249
Eliningaya J. Kweka, Tropical Pesticides Research Institute, P.O.Box 3024, Arusha, [email protected], tel:+255 754 368748
Guiyun Yan, Program in Public Health,University of California, Irvine3501 Hewitt Hall, Irvine, California 92697, [email protected], tel:+1(949)824-0175
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Thomas M. Gilbreath III
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Land Use Change and the Microbial Ecology of Anopheles gambiae
Thomas M. Gilbreath III1, Guofa Zhou
1, Eliningaya J. Kweka
2, and Guiyun Yan
1
1Program in Public Health, University of California, Irvine, CA 92697, USA
2Tropical Pesticides Research Institute, Arusha, Tanzania
Recent studies have suggested that land use changes, such as deforestation, strongly
enhance the productivity of malaria vectors, and thus malaria transmission. However, the
mechanism for habitat productivity enhancement by deforestation is not clear. The present study
conducted a metagenomics analysis of Anopheles gambiae mosquitoes to determine the impacts
of deforestation on bacterial and algal diversity of the larval habitats and subsequent habitat
productivity. The metagenomic analysis was based on high-throughput next-generation
pyrosequencing of microbial 16S and 23S DNA directly extracted from field-collected water and
mosquito specimens to provide a comprehensive view of microbial community diversity. Results
of habitat productivity in different land use scenarios demonstrated a significant effect of land
cover on larval development and adult mosquito productivity. Metagenomic analysis of algal
communities by pyrosequencing of 23S DNA revealed a selective feeding on green algae species
(Chlorophyta), which were far more abundant in the larval gut when compared to the available
potential resources. Quantitative PCR demonstrated that although shaded habitats had higher
bacterial abundance, larval development was poor in these habitats, whereas in sunlit habitats,
bacterial abundance was lower, but larval survivorship was higher and development time was
faster, suggesting algae is a more important food resource for An. gambiae larvae relative to
bacteria. This study provided strong evidence that algal community shifts resulting from
deforestation enhanced habitat productivity for An. gambiae mosquitoes. The implications of
microbial community dynamics in the scope of malaria vector control are discussed.
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Introduction
Over the past three decades, as a result of increasing population and agricultural
development, the western Kenya highlands have undergone considerable environmental changes
(Himeidan and Kweka 2012). Environmental changes, such as temperature, humidity and
habitat availability can significantly affect mosquito abundance, which in turn affect malaria
transmission intensity (Minakawa, Munga et al. 2005). Past studies have suggested that land use
changes, such as deforestation, strongly enhance the productivity of malaria vectors, and thus
malaria transmission (Walsh, Molyneux et al. 1993). This is because deforestation exposes
aquatic habitats to sunlight, resulting in increased water temperatures . Further, exposure to
sunlight may induce changes in the microbial communities that mosquito larvae use for nutrition.
However, there is little knowledge of how microbial communities in aquatic habitats are
regulated by exposure to sunlight and organic nutrients, and how mosquito larvae respond to
microbial community changes in larval habitats (Merritt, Craig et al. 1992; Merritt, Dadd et al.
1992).
The larval ecology of Anopheles gambiae sensu stricto (An. gambiae), the primary
malaria vector in sub-Saharan Africa, remains poorly understood with respect to feeding
preferences and behaviors (Merritt, Dadd et al. 1992); however, it is known that variability in
aquatic habitat characteristics contributes to significant differences in larval abundance
(Minakawa, Munga et al. 2005). Several studies have demonstrated that larval development is
density dependent, suggesting that available food resources may also play a role in regulating
larval habitat productivity (Gimnig, Ombok et al. 2002; Kweka, Zhou et al. 2012). Previous
work has demonstrated the importance of algae in the growth and development of larvae
(Coggeshall 1926; Howland 1930; Kaufman, Wanja et al. 2006), and others have also shown a
reduction in algal abundance in the presence of larvae (Gimnig, Ombok et al. 2002).
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Associations between larvae and green algae species (Chlorophyta; Chlorophyceae) have also
been observed (Tuno, Githeko et al. 2006). The surface microlayers (SuM) of aquatic habitats
typically accumulate high numbers of bacteria, algae, and dissolved organic matter (Merritt,
Dadd et al. 1992). Mosquito larvae of the genus Anopheles are typically oriented horizontally
along the air to water interface, and they do the majority of their feeding in the surface
microlayer of their aquatic habitats (Merritt, Craig et al. 1992).
The current study examined how chemical and physical characteristics of larval habitats
under variable canopy coverage affect larval habitat productivity, and assessed the importance of
canopy coverage independent of temperature. The mechanisms of larval population regulation
have recently received renewed interest (Himeidan and Kweka 2012). We hypothesize that An.
gambiae are largely regulated by physical habitat characteristics and the resultant availability of
nutritional resources. We utilized two experiments to further test the hypothesis that reduced
canopy coverage, with or without variation in temperature, would lead to increased larval habitat
productivity due to the resulting increase of photosynthetic microbes that An. gambiae use for
food. A third experiment was conducted to test the effects of food addition in all experimental
treatments. One important technique we used was the metagenomic analysis of the larval habitats
and An. gambiae mosquito larvae, which offers a comprehensive view of microbial communities
(Wooley, Godzik et al. 2010; Wang, Gilbreath et al. 2011). This approach was based on the high-
throughput next-generation pyrosequencing of microbial 16S and 23S DNA extracted directly
from field-collected water and mosquito specimens, thus avoiding the bias associated with
inability to culture some bacteria or algae required by the traditional methods for detecting and
identifying microbes (Wooley, Godzik et al. 2010). The metagenomics approach provides a more
powerful tool to test the hypothesis that larval gut contents would demonstrate preferential
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feeding by larvae upon the photosynthetic microbes in the surface microlayer of their aquatic
habitats.
Methods
We established an array of artificial habitats, or microcosms, in Iguhu, Kakamega
District, western Kenyan (elevation 1,500m). Three sub-sites were chosen for deforested, semi-
forested and forested habitats (un-shaded, partially shaded and heavily shaded, respectively).
Microcosms for experiment one were constructed using 40cm diameter washtubs, and
approximately two liters of top-soil from the forest edge were added to each. Eight microcosms
were distributed at each sub-site. An. gambiae eggs were transported to the study site and
hatched onsite. One hundred first instar larvae were added into each microcosm. Habitats were
monitored for survivorship and life table data (instar proportions) daily, and adult wing length
was measured. Temperature was logged hourly throughout the duration of the experiment using
HOBO TidbiT water temperature loggers (Onset Computer Corporation, Bourne, MA, USA).
Pooled 50 ml surface water samples (< 2 mm) were taken weekly from each sub-site in triplicate
using a needle and syringe, centrifuged at 10,000 g for 20 minutes, and the resultant pellet was
used for DNA extraction. For each canopy coverage level, triplicate samples of eight larval guts
were removed under a dissecting microscope using sterilized dissection devices.
Temperature-controlled microcosms were constructed using perforated baskets lined with
thin (1 mil) polyethylene plastic sheeting to allow for maximum heat exchange between
microcosms and pools. Replicates were assigned shade or sunlit treatment to mimic forested and
deforested habitats. Shades were constructed with wooden frames and 1.5 mil opaque black
plastic, All microcosms were placed into approximately 4 x 2 x 0.5 meter pools that were filled
with stream water to match the water level in the microcosms.
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. DNA was extracted from larval guts and water sample pellets using the UltraClean Soil
DNA Extraction Kit (Mo Bio Laboratories, Carlsbad, CA, USA). To characterize the bacterial
communities, the variable 16S rDNA region V1-3 was amplified. Algal communities were
characterized using the Domain V of the 23S plastid rRNA gene (Sherwood, Chan et al. 2008).
16S PCR, 23S PCR and pyrosequencing were conducted at Research and Testing Laboratory
(Lubbock, TX) using the Roche Titanium 454 FLX pyrosequencing platform as described
previously (Callaway, Dowd et al. 2010). Sequences were deposited in the NCBI sequence reads
archive (accession number SRA051793). The Ribosomal Database Project (RDP) classifier was
used to assign taxonomic rank with a confidence threshold of 80%
(http://rdp.cme.msu.edu/classifier/classifier.jsp) (Wang, Garrity et al. 2007). We used BLAST
searches to match algal sequences from water and gut samples with known organisms in the
National Center for Biotechnology Information database.
Relative ratios of bacteria were determined with qPCR using bacteria universal
quantitative primers on triplicate samples from the water surface microlayer of habitats. Serial
dilutions of known concentrations of Pseudomonas aeruginosa DNA were used to generate
reference samples in order to determine relative abundance of bacteria as done previously
(Dowd, Hanson et al. 2010).
Food supplementation with approximately 100mg of Tetramin Fish Food (Spectrum
Brands, Inc., Madison, WI, USA) was added daily to eight additional replicates of all treatments
in experiment one and two.
For all experiments, differences in An. gambiae larval survivorship, larval development
time and adult wing length among habitats were compared using analysis of variance (ANOVA)
(JMP Statistical Discovery Software, version 5.1; SAS Institute, Cary, NC). Stepwise
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regression was used to analyze the effects of environmental variables on larval survivorship and
development time, and indirect effects were analyzed using path analysis and performed by using
SPSS Amos (Amos Development Corporation, Meadville, PA 16335, USA). For pyrosequencing
data, bacterial species richness and diversity analyses were conducted using the program
MOTHUR (Schloss, Westcott et al. 2009). We used MOTHUR to generate matrices of genetic
distance and group sequences into operational taxonomic units (OTUs) For each treatment we
used MOTHUR to calculate Chao1 estimates of richness and ACE estimates of diversity
(Wooley, Godzik et al. 2010).
Results/Discussion
The current study examined the relationship between habitat vector productivity,
microbial food resources and deforestation. Bacterial and algal communities are dependent on
temperature in water and soil, but drastic changes in microbial communities, particularly
photosynthetic microbes, also result from sunlight proliferation to the habitat surface as a
consequence of disforestation. The semi-natural, temperature and light variable treatments
confirm previous findings that larval success is limited by canopy coverage (Afrane, Zhou et al.
2007). Temperature-controlled experiments were designed to control water temperatures while
subjecting habitats to variable sunlight levels. Despite temperatures conducive to high larval
survivorship in the shaded, temperature-controlled habitats, pupation rates were low, presumably
due to the lack of available food resources in the sunlight poor environment (Figure 2). These
conclusions are further supported by the results of food supplementation. When shaded habitats
were supplemented with food, larval performance was nearly identical when compared to food
supplemented sunlit habitats (Figure 2). This study did not examine oviposition preference of An.
gambiae mosquitoes for open sunlit habitats, but it provided strong evidence for enhanced larval
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development and survivorship as a direct result of microbial community changes in sunlit
habitats.
Of these microbial communities, bacterial and algal communities are thought to be of
most importance (Merritt, Dadd et al. 1992; Kaufman, Wanja et al. 2006). While foraging on
biofilms has been shown to be of particular importance to Aedes albopictus and Ae. aegypti ,
algal abundance has been suggested as an important factor for anophelines (Kaufman, Wanja et
al. 2006; Garros, Ngugi et al. 2008). The current study examined the relative abundance of algae
and bacteria in relation to larval success. Relative bacterial abundance indicated that bacterial
abundance increased with increasing canopy coverage. Even though shaded habitats in
temperature-controlled experiments had higher bacterial abundance, larval development was
poor in these habitats. Further, in sunlit habitats, where lower bacterial abundance was
observed, larval survivorship was higher and development time was faster. Given that these
results were from temperature-controlled microcosms, our study strongly suggest that algae, not
bacteria, is the most important food source for An. gambiae larvae, and habitat productivity is
mostly limited by algal concentration of larval habitats.
The Proteobacteria were detected in highest abundance in all samples from both the water
SuM and LGC; however, a notable shift in Proteobacteria class representation was observed. The
Enterobacteriaceae (Gammaproteobacteria) were shown to be effective colonizers of the larval
gut, and previous work has demonstrated that the adult gut selectively favors the colonization of
Enterobacteriaceae (Wang, Gilbreath et al. 2011) (Figure 1).
The mechanisms for differential ingestion of certain microbial groups by An. gambiae
larvae are not clear. One potential mechanism is larval behavioral modification in response to
resource availability. For example, it has been shown that An. quadrimaculatus larvae employ a
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suite of behaviors to discriminate between available food resources on or near the surface and in
the water column (Merritt, Craig et al. 1992). Feeding intensity can also be up-regulated by the
presence of phagostimulants produced by microbial sources (Dadd 1970).
Productive anopheline habitats have long been strongly correlated with sunlight and the
presence of algae (Coggeshall 1926; Howland 1930; Gimnig, Ombok et al. 2002; Tuno, Githeko
et al. 2006). Further, Tuno et al. found an association between the green alga, Rhopalosolen
species (Chlorophyta) and An. gambiae abundance and body size (Tuno, Githeko et al. 2006).
Although no Chlorophyta sequences were obtained from the forest water samples, the gut
contents of larvae reared in the forest were dominated by chlorophytes (≈ 84%) (Figure 1). This
distinction is likely due to the very low abundance of chlorophytes in the habitats and further
reduction by larvae. Alternatively, larvae may be employing atypical foraging techniques (i.e.,
scraping, shredding) in the forested habitats with low algal growth. Surprisingly, diatoms rather
than chlorophytes dominated the LGC from the deforested area (Figure 1). This is likely due to
the abundance of photosynthetic food resources in the deforested site and a buildup of diatoms
that are more resistant to digestion in the mosquito gut due to their characteristic cell wall
composed of recalcitrant silica .
Approximately 80% of bacterial sequences were identified to species based on sequence
similarity analysis; however, algae were only identified to the family level (48%) due to the
limited number of sequences available in GenBank. We did not determine the contribution of
individual microbial species to nutrition of mosquito larvae, but the mosquito developmental
data from the temperature-controlled experiments is consistent with the general findings of the
pyrosequencing data: habitats lacking algal growth largely do not support larvae to adulthood.
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These results demonstrate that sunlight proliferation to habitats and the resultant growth
of photosynthetic microbes are critical to the success of Anopheles gambiae mosquitoes. Further,
this work suggests that larval habitat productivity is up-regulated by the eutrophication of aquatic
habitats associated with agriculture. The combined effect of sunlight proliferation due to canopy
removal and the use of fertilizers contribute to habitat eutrophication and algal blooms that will
be beneficial to larval populations. This study enhances our ability to identify or predict aquatic
habitats suitable for malaria vector production and thus facilitates mosquito larval control
targeted at the most productive habitats.
ACKNOWLEDGEMENTS
We thank Anne Vardo-Zalik and Ming-Cheih Lee for helpful discussion and expertise, and the
two anonymous reviewers for constructive comments. This work was supported by grants from
the National Institutes of Health (R01 AI050243 and D43 TW001505)
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FIGURE 1. Bacterial (A) and algal (B) diversity at the phylum level revealed by
pyrosequencing. Major bacterial phyla are presented and remaining taxa and unclassified
bacteria are pooled and referred to as Other. SuM: water surface microlayer; LGC: third-instar
larval gut contents.
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FIGURE 2. Chlorophyll a concentration (A) and pupation rates (B) of Anopheles gambiae in
temperature controlled microcosms, with and without food supplementation. Food
supplementation in shaded microcosms resulted in nearly identical pupation rates to sunlit
microcosms (P = 0.93). Food supplementation also resulted in increased chlorophyll a and larval
pupation rates in all semi-natural treatments.
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