Thorsellia Anophelis Is the Dominant Bacterium in a Kenyan Population of Adult Anopheles Gambiae Mosquitoes
Total Page：16
File Type：pdf, Size：1020Kb
The ISME Journal (2008) 2, 74–82 & 2008 International Society for Microbial Ecology All rights reserved 1751-7362/08 $30.00 www.nature.com/ismej ORIGINAL ARTICLE Thorsellia anophelis is the dominant bacterium in a Kenyan population of adult Anopheles gambiae mosquitoes Aurelio M Briones1, Josephat Shililu2, John Githure2, Robert Novak3 and Lutgarde Raskin1 1Department of Civil and Environmental Engineering, University of Michigan, Ann Arbor, MI, USA; 2International Centre of Insect Physiology and Ecology, Nairobi, Kenya and 3Illinois Natural History Survey, Champaign, IL, USA Anopheles gambiae mosquitoes are not known to harbor endosymbiotic bacteria. Here we show, using nucleic acid-based methods, that 16S rRNA gene sequences specific to a recently described mosquito midgut bacterium, Thorsellia anophelis, is predominant in the midgut of adult An. gambiae s.l. mosquitoes captured in residences in central Kenya, and also occurs in the aquatic rice paddy environment nearby. PCR consistently detected T. anophelis in the surface microlayer of rice paddies, which is also consistent with the surface-feeding behavior of A. gambiae s.l. larvae. Phylogenetic analysis of cloned environmental 16S rRNA genes identified four major Thorsellia lineages, which are closely affiliated to an insect endosymbiont of the genus Arsenophonus. Physiological characterizations support the hypothesis that T. anophelis is well adapted to the female anopheline midgut by utilizing blood and tolerating the alkaline conditions in this environment. The results suggest that aquatically derived bacteria such as T. anophelis can persist through mosquito metamorphosis and become well-established in the adult mosquito midgut. The ISME Journal (2008) 2, 74–82; doi:10.1038/ismej.2007.95; published online 1 November 2007 Subject Category: microbe–microbe and microbe–host interactions Keywords: Anopheles; Kenya; paratransgenics; rice paddy; thorsellia Introduction a symbiotic or consistent association between Anopheles gambiae s.l. and specific species of The life cycle of the malaria parasite (Plasmodium bacteria has not been described. Among other spp) involves critical steps within the mosquito Anopheles species, An. stephensi has been reported midgut, an environment it shares with bac- to stably associate with bacteria of the genus Asaia teria. Various physical (for example, membranes, (Favia et al., 2007). Such a relationship would make receptors), biochemical (for example, proteases, use of genetically transformed bacteria in para- chitinases) and physiological (for example, immu- transgenesis more efficient (Beard et al., 2002). nological factors) barriers protect the insect against Paratransgenics refers to the genetic transformation the parasite (for reviews see Sinden, 2002; Meister of a bacterium, typically a vector endosymbiont, to et al., 2004), but the largest losses in parasite titer produce antiparasitic molecules, thus inhibiting occur when Plasmodium zygotes develop into parasite development (Conte, 1997; Beard et al., ookinetes within the adult mosquito midgut. During 2002). Since Anopheles mosquitoes are currently this transition, the parasite may undergo more than considered a dead end host for midgut bacteria that two orders of magnitude loss in abundance (Vaugh- are believed to ‘perish when the adult mosquito an et al., 1994). Although the negative impact of dies’ (Riehle and Jacobs-Lorena, 2005), the current midgut bacteria on parasite development has been paradigm for paratransgenesis in Anopheles focuses observed in the past (Pumpuni et al., 1993; Low- on bacteria that opportunistically infect adult enberger et al., 1999; Gonzalez-Ceron et al., 2003), mosquitoes (Lindh et al., 2006; Favia et al., 2007; Riehle et al., 2007). The most significant perturbation affecting mos- Correspondence: AM Briones, Department of Civil and Environ- quito midgut microbial communities occurs during mental Engineering, University of Michigan, 1351 Beal Ave., 3 metamorphosis from pupae to adults. For bacteria EWRE Bldg, Ann Arbor, MI 48109-2125, USA. E-mail: [email protected] inhabiting the midgut, the process has been Received 19 June 2007; revised 1 October 2007; accepted 3 characterized as similar to ‘gut sterilization’ October 2007; published online 1 November 2007 (Moll et al., 2001), which, if correct, would imply Thorsellia in Anopheles gambiae s.l. midguts AM Briones et al 75 that environmentally derived bacteria that are prior to sampling. Surface microlayer (SML) present in larval midguts (through feeding) samples of two randomly selected plots from each are greatly attenuated or are absent in adult midguts. row (designated plots A and B) were collected using It should be stressed, however, that microbial a stainless steel mesh screen measuring 400 cm2, studies of the midgut of Anopheles are scarce, and with a mesh size of 1.25 and 0.36 mm wire diameter have depended mainly on traditional culture-based as described previously (Agogue et al., 2004). techniques (Pumpuni et al., 1996; Straif et al., 1998; Mosquito larvae and bulk water from four plots Gonzalez-Ceron et al., 2003; Lindh et al., 2005). (plots A, B, C and D; two plots per row) were One study has coupled culture- and PCR-based sampled with a 350 ml plastic dipper, using stan- approaches to characterize An. gambiae s.l. midgut dard dipping techniques (Service, 1993). Since the bacteria (Lindh et al., 2005). Therefore, the applica- larval abundance was low during the time of tion of other nucleic acid-based tools, such as sampling, mosquito larvae from the remaining 12 denaturing gradient gel electrophoresis (DGGE) plots were also collected and pooled according to (Muyzer et al., 1993), to study these systems is the row. Among the pooled water samples from each timely. plot, 300 ml (from 30 pooled dips of SML water) and In this study, we provide evidence from PCR, 500 ml (from 20 pooled dips of bulk water) aliquots DGGE and clone library analyses that adult field- were placed in sterile Nalgene bottles (Nalge Nunc captured An. gambiae s.l. mosquitoes in a Kenyan International, Rochester, NY, USA) and transported village are consistently associated with a Thorsellia on ice immediately to the laboratory. All water and anophelis lineage that was also detected in the larval samples were processed within 24 h of surface microlayer of rice paddies. Unlike most collection. Female An. gambiae s.l. fourth instar members of the Enterobacteriaceae, this bacterium larvae were selected morphologically (Gillies and grows slowly in rich media, but is well adapted to Coetzee, 1987) and rinsed three times in filter- the anopheline midgut because of its ability sterilized phosphate-buffered saline (137 mM so- to utilize blood to enhance growth and its tolerance dium chloride, 2.7 mM potassium chloride and to alkaline conditions in the anopheline midgut. 10 mM sodium phosphate dibasic/potassium phosphate monobasic, pH 7.4) prior to dissection. The larvae were dissected using aseptic techniques, Materials and methods with the aid of a dissecting microscope (Zeiss Stemi 1000; Carl Zeiss Inc., Jena, Germany). Each midgut Study site and sample collection sample subjected to DNA extraction consisted of 30 The study site is situated within the Mwea Irrigation larval midguts pooled together in sterile phosphate- and Agricultural Development experimental station, buffered saline and corresponded to larvae collected located approximately 100 km northeast of Nairobi, from two adjacent rows of plots. Kenya (371 200 E and 01 400 S), at an elevation of Indoor resting mosquitoes from two houses (de- 1159 m above sea level. The site falls within a region signated house 1 and house 2) approximately 100 m of low stable transmission of the malaria parasite, apart were captured in the morning hours starting which infects around 20% of the human population around 0700 hours using the pyrethrum spray (Mutero et al., 2000). A detailed description of the collection method (Service, 1993). Immediately after site has been reported elsewhere (Muturi et al., collection, mosquitoes were transported to the 2006). The experimental plots were laid out in a laboratory and adult female An. gambiae s.l. 0.4 ha area, which is situated within 100 m of mosquitoes were selected based on morphology Kariua, a village inhabited mostly by subsistence- (Gillies and Coetzee, 1987). The samples were stored level farmers living in houses with walls at À20 1C in phosphate-buffered saline solution constructed of mud or wood. The experimental containing 30% glycerol and 1% Tween 80. Dissec- plots are arranged in eight blocks (rows) subdivided tion and midgut collection of adult mosquitoes was into eight plots (6.3 Â 3.15 m), with each block carried out as described above. Each pooled sample hydrologically isolated using unidirectional inflow of 30 midguts corresponded to adult mosquitoes and outflow canals. Among the 64 plots, two collected from one house. Greater than 50% of the adjacent rows (designated rows 1 and 2) were set extracted midguts were derived from blood-fed aside for the activities described in this study. All anophelines. the plots were exposed to natural colonization by mosquitoes. Previous fieldwork has shown that the most abundant anopheline species within Mwea DNA extraction, Bacterial PCR and DGGE conditions Irrigation and Agricultural Development rice pad- DNA from all samples was extracted using the dies is An. arabiensis (a sub-species of An. gambiae UltraClean soil DNA isolation kit (MO BIO Labora- s.l.) (Mutero et al., 2000).